Method for the Treatment of a Solid Tumour

ABSTRACT

The present invention relates generally to a method of treating a neoplastic condition and to agents useful for same. More particularly, the present invention is directed to a method of facilitating the treatment of a solid tumour in a localised manner via the co-administration of particulate material and a cellular toxin. The method of the present invention is useful in a range of therapeutic treatments including the treatment of primary and metastatic tumours.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/853,069, filed Dec. 22, 2017, which is a continuation of U.S.application Ser. No. 14/112,673, filed Dec. 4, 2013, which is a NationalStage Application under 35 U.S.C. 371 of co-pending PCT applicationPCT/AU2012/000414 designating the United States and filed Apr. 20, 2012;which claims the benefit of AU application number 2012900480 and filedFeb. 9, 2012; which claims the benefit of AU application number2011901495 and filed Apr. 20, 2011; which claims the benefit of USapplication No. 61/477,382 and filed Apr. 20, 2011.

FIELD OF THE INVENTION

The present invention relates generally to a method of treating aneoplastic condition and to agents useful for same. More particularly,the present invention is directed to a method of facilitating thetreatment of a solid tumour in a localised manner via theco-administration of particulate material and a cellular toxin. Themethod of the present invention is useful in a range of therapeutictreatments including the treatment of primary and metastatic tumours.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by the author inthis specification are collected alphabetically at the end of thedescription.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

Malignant tumours, or cancers, grow in an uncontrolled manner, invadenormal tissues, and often metastasize and grow at sites distant from thetissue of origin. In general, cancers are derived from one or only a fewnormal cells that have undergone a poorly understood process calledmalignant transformation. Cancers can arise from almost any tissue inthe body. Those derived from epithelial cells, called carcinomas, arethe most common kinds of cancers. Sarcomas are malignant tumours ofmesenchymal tissues, arising from cells such as fibroblasts, musclecells, and fat cells. Solid malignant tumours of lymphoid tissues arecalled lymphomas, and marrow and blood-borne malignant tumours oflymphocytes and other hematopoietic cells are called leukaemias.

Cancer is one of the three leading causes of death in industrialisednations. As treatments for infectious diseases and the prevention ofcardiovascular disease continues to improve, and the average lifeexpectancy increases, cancer is likely to become the most common fataldisease in these countries. Therefore, successfully treating cancerrequires that all the malignant cells be removed or destroyed withoutkilling the patient. An ideal way to achieve this would be to induce animmune response against the tumour that would discriminate between thecells of the tumour and their normal cellular counterparts. However,immunological approaches to the treatment of cancer have been attemptedfor over a century with unsustainable results.

Accordingly, current methods of treating cancer continue to follow thelong used protocol of surgical excision (if possible) followed byradiotherapy and/or chemotherapy, if necessary. The success rate of thisrather crude form of treatment is extremely variable but generallydecreases significantly as the tumour becomes more advanced andmetastasises. Further, these treatments are associated with severe sideeffects including disfigurement and scarring from surgery (e.g.mastectomy or limb amputation), severe nausea and vomiting fromchemotherapy, and most significantly, the damage to normal tissues suchas the hair follicles, gut and bone marrow which is induced as a resultof the relatively non-specific targeting mechanism of the toxic drugswhich form part of most cancer treatments and is a major limiting factorfor dosage

Still further, common chemotherapy drugs do not significantly penetrateinto tissue further than about 70 microns from the blood supply (Primeauet al. Clin. Canc. Res. 2005, 11:8782-8788; Minchinton et al. Nat. Rev.Cancer 2006, 6:583-592). The rapid growth and poor vascular developmentof most solid tumours puts many tumour cells well beyond the capacity ofthe drugs to penetrate the tissue. Critically, many cells experiencesub-lethal doses, allowing them to survive and to develop drugresistance.

Solid tumours cause the greatest number of deaths from cancer and mainlycomprise tumours of the linings of the bronchial tree and the alimentarytract that are known as carcinomas. In the year 2000 in Australia,cancer accounted for 30% of male deaths and 25% of female deaths (Cancerin Australia 2000, 2003) and it accounted for 24% of male and 22% offemale deaths in the US in year 2001 (Arias et al. 2003, National VitalStatistics Reports 52:111-115). Solid tumours are not usually curableonce they have spread or ‘metastasised’ throughout the body. Theprognosis of metastatic solid tumours has improved only marginally inthe last 50 years. The best chance for the cure of a solid tumourremains in the use of local treatments such as surgery and/orradiotherapy when the solid tumour is localised to its originatinglining and has not spread either to the lymph nodes that drain thetumour or elsewhere. Nonetheless, even at this early stage, andparticularly if the tumour has spread to the draining lymph nodes,microscopic deposits of cancer known as micrometastases may have alreadyspread throughout the body and will subsequently lead to the death ofthe patient. In this sense, cancer is a systemic disease that requiressystemically administered treatments. Of the patients who receivesurgery and/or radiotherapy as definitive local treatment for theirprimary tumour and who have micrometastases, a minor proportion may becured or at least achieve a durable remission from cancer by theaddition of adjuvant systemic treatments such as cytotoxic chemotherapyor hormones.

Conventionally, solid cancer has been treated locally with surgeryand/or radiotherapy, and during its metastatic stage with systemicallyadministered cytotoxic drugs, which often interfere with the cell cycleof both normal and malignant cells. The relative selectivity of thisapproach for the treatment of malignant tissues is based to some extenton the more rapid recovery of normal tissues from cytotoxic drug damage.More recently, the targeted therapy of cancer has aimed to improve thetherapeutic ratio of cancer treatment by enhancing its specificityand/or precision of delivery to malignant tissues while minimisingadverse consequences to normal non-malignant tissues. Two of the majorclasses of targeted therapy are (i) the small molecule inhibitors suchas the tyrosine kinase inhibitors imatinib mesylate (Glivec®), gefitinib(Iressa®) and erlotinib (Tarceva®), and (ii) the monoclonal antibodies(mAb) such as rituximab (Mabthera®) and trastuzumab (Herceptin®).

In parallel to the development of targeted therapies, combining at leasttwo conventional anti-cancer treatments such as chemotherapy andradiotherapy in novel ways has been another approach to the developmentof cancer therapeutics. By exploiting synergistic interactions betweenthe different modalities of treatment, combined modality treatment seeksto improve treatment efficacy so that the therapeutic ratio for thecombined treatment is superior to that for each of the individualtreatments.

Combined modality treatment using external beam radiation andradiosensitising chemotherapeutic drugs such as 5-fluorouracil andcisplatin (chemoradiotherapy) has improved survival in a number of solidtumours such as those of head and neck, lung, oesophagus, stomach,pancreas and rectum because of both improved local tumour control andreduced rates of distant failure (T S Lawrence. Oncology (Huntington)17:23-28, 2003). Although radiosensitising drugs increase tumourresponse, they also increase toxicity to adjacent normal tissues, whichis especially true of the potent new generation radiosensitisers,gemcitabine and docetaxel. However, decreasing the radiation volumeallows cytotoxic doses of gemcitabine to be better tolerated clinically(Lawrence 2003, supra). Chemoradiotherapy may overcome mutuallyreinforcing resistance mechanisms, which may only manifest in vivo.

Radioimmunotherapy (RIT) is a systemic treatment that takes advantage ofthe specificity and avidity of the antigen-antibody interaction todeliver lethal doses of radiation to cells that bear the target antigen.Radio-isotopes that emit β-particles (e.g. ¹³¹lodine, ⁹⁰Yttrium,188Rhenium, and ⁶⁷Copper) are usually used to label monoclonalantibodies (mAb) for therapeutic applications. The energy from□-radiation is released at relatively low intensity over distancesmeasured in millimeters (Waldmann, Science 252:1657-1662, 1991; Benderet al., Cancer Research 52:121-126, 1992; O'Donoghue et al. Journal ofNuclear Medicine 36:1902-1909, 1995; Griffiths et al. InternationalJournal of Cancer 81:985-992, 1999). Thus, high-energy □-emitters suchas ⁹⁰Yttrium are useful for the treatment of larger and heterogeneoussolid tumours (Liu et al. Bioconjugate Chemistry 12:7-34, 2001).Research interest in radioimmunotherapy has been reawakened because inspite of the low radiation doses delivered, significant and unexpectedbiological effects of RIT upon surrounding host cells have been observed(Xue et al. Proceedings of the National Academy of Sciences of theUnited States of America 99:13765-13770, 2002). Furthermore, the lowerbut biologically effective dose of radiation delivered by RIT hadgreater cytocidal effects than a larger dose of radiation conveyed asexternal beam radiotherapy (Dadachova et al., PNAS 101:14865-14870,2004). Nonetheless, the efficiency of RIT as a treatment for solidtumours may be hampered by the low penetration of antibody through thetissue barriers that surround the target antigen in the tumour, whichwill consequently extend circulatory half life of the antibody(Britz-Cunningham et al. Journal of Nuclear Medicine 44:1945-1961,2003). Furthermore, RIT is often impeded by the heterogeneity of thetarget antigen's expression within the tumour. Thus, although RITaffords molecular targeting of tumour cells, the major limitation of RITremains the toxicity that may result from large doses of radiation thatare delivered systemically in order to achieve sufficient targeting(Britz-Cunningham et al. 2003, supra; Christiansen et al. MolecularCancer Therapy 3:1493-1501, 2004). Altogether, a useful therapeuticindex using RIT has proven difficult to achieve clinically (Sellers etal. Journal of Clinical Investigation 104:1655-1661, 1999).

Tumour associated antigens, which would allow differential targeting oftumours, while sparing normal cells, have also been the focus of cancerresearch. Although abundant ubiquitous antigens may provide a moreconcentrated and accessible target for RIT, studies adopting this havebeen extremely limited.

The development of nanoparticle technology was also hailed as anexciting new frontier in terms of the development of new and effectivecancer treatments. However, although previous attempts at usingparticulate material, such as nanoparticles, to target tumours foreither diagnostic or therapeutic purposes have been extensive, in thecontext of therapeutics there has, disappointingly, been minimalsuccess. With diagnostics, relatively shallow penetration of theparticles into the tumour has been sufficient to achieve the objectiveof visualising the tumour. However, in terms of the delivery of atherapeutic agent, such shallow penetration has not been sufficient toeffectively deliver the agent throughout the tumour, in particular tothe interior of the tumour, as is required if total tumour destructionis to be achieved. In relation to therapeutics, specifically,conjugation of particles to a wide variety of different materials has sofar failed to live up to the promise of achieving effective tumourpenetration, this being an essential prerequisite for a therapeutic tohave any chance of effectiveness.

Significant effort has also been made to take advantage of the enhancedpermeability and retention (EPR) effect of tumours as a means to developan effective therapeutic. Without limiting the present invention to anyone theory or mode of action, this is a well described phenomenon basedon the notion that certain sizes of molecules, typically liposomes ormacromolecular drugs, tend to preferentially accumulate in tumourtissue. The general explanation for this phenomenon is that, in orderfor tumour cells to grow quickly, they must stimulate the production ofblood vessels. VEGF and other growth factors are involved in cancerangiogenesis. Tumour cell aggregates of sizes as small as 150-200 μmbecome dependent on blood supply carried by neovasculature for theirnutritional and oxygen supply. These newly formed tumour vessels areusually abnormal in form and architecture. They comprise poorly-aligneddefective endothelial cells with wide fenestrations, lacking a smoothmuscle layer, or innervation with a wider lumen, and impaired functionalreceptors for angiotensin II. Furthermore, tumour tissues usually lackeffective lymphatic drainage. All these factors will lead to abnormalmolecular and fluid transport dynamics, especially for macromoleculardrugs. Accordingly, it has been thought that one way to achieveselective drug targeting to solid tumours is to exploit theseabnormalities of tumour vasculature in terms of active and selectivedelivery of anticancer drugs to tumour tissues, notably defining the EPReffect of macromolecular drugs in solid tumours. Due to their largemolecular size, nanosized macromolecular anticancer drugs administeredintravenously escape renal clearance. Often they cannot penetrate thetight endothelial junctions of normal blood vessels, but they canextravasate in tumour vasculature and become trapped in the tumourvicinity. Nevertheless, the EPR effect has not been efficiently orsuccessfully harnessed.

Various nanoparticles have been designed which are directed to achievingefficient cellular endocytosis. However, even if this is achievable, theissue of tissue penetration is still a separate one which, to date, hasnot been successfully overcome. The general notion of the use of ananoparticle as a vector for delivery of a drug is widely discussed inthe literature but, in the absence of achieving deep tumour penetration,is of limited value.

Even where effective tumour distribution of a drug is achieved (bywhatever means) a further problem has been the fact that neoplasticcells within solid tumours can exhibit a slowed metabolism. This meansthat even if a cytotoxic drug penetrates to these cells, if it is noteffectively metabolised it will have a limited impact on the viabilityof the tumour.

Accordingly, there is an urgent and ongoing need to develop improvedsystemic therapies for solid cancers, in particular metastatic cancers.

In work leading up to the present invention it has been determined thatparticulate material which is maintained in a dispersed state by astabiliser is able to achieve deeper penetration into solid tumourmodels than has previously been achievable using nanoparticletechnology. This has enabled the development of an effective means fortreating solid tumours, both primary and metastatic, based on theco-administration of a cellular toxin with the particulate material. Byeither sequentially or simultaneously delivering this toxin, deeperpenetration and therefore more extensive cellular exposure to the toxinis achieved. By virtue of the less effective reticuloendothelialclearance which is associated with tumours, a form of targeted treatmentis effectively achieved. Still further, it has been observed that thetoxin uptake by tumours penetrated by the particles of the presentinvention is effective, suggesting upregulation of tumour cellmetabolism. Accordingly, the method of the present invention provides ameans for achieving a more effective localised delivery and uptake of acellular toxin to a tumour and its metastases in a manner which ischaracterised by significantly improved outcomes and/or reduced sideeffects relative to those which would normally be expected in thecontext of conventional treatment of an equivalent type of tumour. Thisis an extremely significant development since current protocols directedto treating metastatic disease are based on the non-targeted systemicdelivery of chemotherapeutic agents.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein, the term “derived from” shall be taken to indicate thata particular integer or group of integers has originated from thespecies specified, but has not necessarily been obtained directly fromthe specified source. Further, as used herein the singular forms of “a”,“and” and “the” include plural referents unless the context clearlydictates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

One aspect of the present invention is directed to a method of treatinga solid tumour in a subject, said method comprising co-administering tosaid subject an effective amount of particulate material and a cellulartoxin for a time and under conditions sufficient to facilitatedistribution of said particulate material and toxin to said tumourwherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a stabiliser; and    -   (ii) said stabiliser comprising an anchoring portion that (a)        anchors the stabiliser to the particulate material, and (b) is        different from the remainder of the stabiliser;        and wherein said particulate material and toxin penetrate said        solid tumour.

For convenience, said particulate material that is maintained in thedispersed state by a stabiliser may herein be referred to as “stabilisedparticulate material”.

In one embodiment, the stabiliser is a steric stabiliser, said stericstabiliser comprising a steric stabilising polymeric segment and ananchoring portion, wherein the steric stabilising polymeric segment isdifferent from the anchoring portion, and wherein the anchoring portionanchors the stabiliser to the particulate material

The method may therefore comprise co-administering to said subject aneffective amount of particulate material and a cellular toxin for a timeand under conditions sufficient to facilitate distribution of saidparticulate material and toxin to said tumour wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a steric stabiliser; and    -   (ii) said steric stabiliser comprising a steric stabilising        polymeric segment and an anchoring portion, wherein the steric        stabilising polymeric segment is different from the anchoring        portion, and wherein the anchoring portion anchors the        stabiliser to the particulate material;        and wherein said particulate material and toxin penetrate said        solid tumour.

In another embodiment, said solid tumour is benign.

In a further embodiment said tumour is malignant.

In yet another embodiment, said anchoring portion is an anchoringpolymeric segment. In that case, said stabiliser comprises an anchoringpolymeric segment, or said steric stabiliser comprises a stericstabilising polymeric segment and an anchoring polymeric segment.

In a further embodiment, said stabiliser comprises an anchoring portion,one or both of the stabiliser or anchoring portion being derived fromone or more ethylenically unsaturated monomers that have beenpolymerised by a living polymerisation technique, wherein the anchoringportion is different from the remainder of the stabiliser, and whereinthe anchoring portion anchors the stabiliser to the particulatematerial. According to this embodiment, the anchoring portion may bereferred to as an anchoring polymeric segment.

In another embodiment, said steric stabiliser comprises a stericstabilising polymeric segment and an anchoring polymeric segment, one orboth of which are derived from one or more ethylenically unsaturatedmonomers that have been polymerised by a living polymerisationtechnique, wherein the steric stabilising polymeric segment is differentfrom the anchoring polymeric segment, and wherein the anchoringpolymeric segment anchors the stabiliser to the particulate material.

In another aspect the present invention provides a method of treating amalignant solid tumour in a subject, said method comprisingco-administering to said subject an effective amount of particulatematerial and a cellular toxin for a time and under conditions sufficientto facilitate distribution of said particulate material and toxin tosaid tumour wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a stabiliser; and    -   (ii) said stabiliser comprising an anchoring portion that (a)        anchors the stabiliser to the particulate material, and (b) is        different from the remainder of the stabiliser;        and wherein said particulate material and toxin penetrate said        solid tumour.

Where the stabiliser is a steric stabiliser comprising a stericstabilising polymeric segment and an anchoring portion, wherein thesteric stabilising polymeric segment is different from the anchoringportion, the method of treating a malignant solid tumour in a subjectcomprises co-administering to said subject an effective amount ofparticulate material and a cellular toxin for a time and underconditions sufficient to facilitate distribution of said particulatematerial and toxin to said tumour wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a steric stabiliser; and    -   (ii) said steric stabiliser comprising a steric stabilising        polymeric segment and an anchoring portion, wherein the steric        stabilising polymeric segment is different from the anchoring        portion, and wherein the anchoring portion anchors the        stabiliser to the particulate material;        and wherein said particulate material and toxin penetrate said        solid tumour.

In one embodiment, said malignant solid tumour is a metastatic malignantsolid tumour. Reference to “metastatic” should be understood as areference to a tumour which either has undergone metastatisation or mayhave undergone metastatisation.

In another embodiment, said malignant solid tumour is a central nervoussystem tumour, retinoblastoma, neuroblastoma, paediatric tumour, headand neck cancer such as squamous cell cancer, breast and prostatecancer, lung cancer, kidney cancers, such as renal cell adenocarcinoma,oesophagogastric cancer, hepatocellular carcinoma, pancreaticobiliaryneoplasia, such as adenocarcinomas and islet cell tumours, colorectalcancer, cervical cancer, anal cancer, uterine or other reproductivetract cancer, urinary tract cancer, such as of the ureter or bladder,germ cell tumour such as a testicular germ cell tumour or ovarian germcell tumour, ovarian cancer, such as an ovarian epithelial cancer,carcinoma of unknown primary, human immunodeficiency associatedmalignancy, such as Kaposi's sarcoma, lymphoma, leukemia, malignantmelanoma, sarcoma, endocrine tumour, such as of the thyroid gland,mesothelioma or other pleural or peritoneal tumour, neuroendocrinetumour or carcinoid tumour.

By “co-administration” is meant that the stabilised particulate materialand the cellular toxin are administered as separate entities in theirown right. In other words, at the time of administration the stabilisedparticulate material and the cellular toxin are not covalently orchemically coupled to each other.

Co-administration of the stabilised particulate material and thecellular toxin in the context of the present invention includes bothsimultaneous and sequential administration. Simultaneous administrationincludes where the stabilised particulate material and the cellulartoxin are present in the same formulation or in two differentformulations, but each are nevertheless administered at substantiallythe same time. In the case of sequential administration, a multi-stepprocedure is used where the stabilised particulate material isadministered in one step and the cellular toxin is administered at adifferent time in a separate step. The cellular toxin may beadministered prior to administration of the stabilised particulatematerial. The time difference between administration of the stabilisedparticulate material and the cellular toxin in sequential administrationcan vary, but will generally range from about 1 minute to about 4 days,for example from about 1 minute to about 2 hours, or from about 1 minuteto about 24 hours, or from about 1 minute to about 12 hours, or fromabout 1 minute to about 6 hours, or from about 1 minute to about 3hours, or from about 1 minute to about 1 hour.

In a sequential administration, the stabilised particulate material willgenerally be administered prior to the cellular toxin.

The particulate material and the cellular toxin may be administered bythe same or different routes.

Without limiting the present invention to any one theory or mode ofaction, once the particulate material has penetrated the tumour,effective penetration of the administered cellular toxin is alsoachieved.

It will be appreciated that it is well within the skills of the personin the art, and in light of the teaching provided herein, to select anddesign an administration protocol for the elements herein described.

In a further aspect there is provided a method of treating a solidtumour in a subject, said method comprising:

-   -   (a) administering to said subject an effective amount of        particulate material and for a time and under conditions        sufficient to facilitate distribution of said particulate        material to said tumour wherein:        -   (i) said particulate material is administered in the form of            a dispersion in a liquid carrier, the particulate material            being maintained in the dispersed state by a stabiliser; and        -   (ii) said stabiliser comprising an anchoring portion            that (a) anchors the stabiliser to the particulate material,            and (b) is different from the remainder of the stabiliser;            and    -   (b) administering to said subject an effective amount of a        cellular toxin subsequently to administration of said        particulate material;        and wherein said particulate material and toxin penetrate said        solid tumour.

Where the stabiliser is a steric stabiliser comprising a stericstabilising polymeric segment and an anchoring portion, wherein thesteric stabilising polymeric segment is different from the anchoringportion, the method of treating a solid tumour in a subject comprises:

-   -   (a) administering to said subject an effective amount of        particulate material and for a time and under conditions        sufficient to facilitate distribution of said particulate        material to said tumour wherein:        -   (i) said particulate material is administered in the form of            a dispersion in a liquid carrier, the particulate material            being maintained in the dispersed state by a steric            stabiliser; and        -   (ii) said steric stabiliser comprising a steric stabilising            polymeric segment and an anchoring portion, wherein the            steric stabilising polymeric segment is different from the            anchoring portion, and wherein the anchoring portion anchors            the stabiliser to the particulate material; and    -   (b) administering to said subject an effective amount of a        cellular toxin subsequently to administration of said        particulate material;        and wherein said particulate material and toxin penetrate said        solid tumour.

In yet another aspect there is provided a method of treating a solidtumour in a subject, said method comprising co-administering to saidsubject an effective amount of particulate material and a cytostatic orcytocidal agent for a time and under conditions sufficient to facilitatedistribution of said particulate material and toxin to said tumour,wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a stabiliser; and    -   (ii) said stabiliser comprising an anchoring portion that (a)        anchors the stabiliser to the particulate material, and (b) is        different from the remainder of the stabiliser; and wherein said        particulate material and said cytostatic or cytocidal agent        penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a stericstabilising polymeric segment and an anchoring portion, wherein thesteric stabilising polymeric segment is different from the anchoringportion, the method of treating a solid tumour in a subject comprisesco-administering to said subject an effective amount of particulatematerial and a cytostatic or cytocidal agent for a time and underconditions sufficient to facilitate distribution of said particulatematerial and toxin to said tumour, wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a steric stabiliser; and    -   (ii) said steric stabiliser comprising a steric stabilising        polymeric segment and an anchoring portion, wherein the steric        stabilising polymeric segment is different from the anchoring        portion, and wherein the anchoring portion anchors the        stabiliser to the particulate material;        and wherein said particulate material and said cytostatic or        cytocidal agent penetrate said solid tumour.

Examples of cytotoxic agents include, but are not limited to,Actinomycin D, Adriamycin, Arsenic Trioxide, Asparaginase, Bleomycin,Busulfan, Camptosar, Carboplatinum, Carmustine, Chlorambucil, Cisplatin,Corticosteroids, Colicheamicin, Cyclophosphamide, Daunorubicin,Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine,Fluorouracil, Gemcitabina, Gemcitabine, Gemzar, Hydroxyurea, Idarubicin,Ifosfamide, Irinotecan, Lomustine, Melphalan, Mercaptomurine,Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel,Platinol, Platinex, Procarbizine, Raltitrexeel, Rixin, Steroids,Streptozocin, Taxol, Taxotere, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulfan, Trihydrate, Vinblastine, Vincristine, Vindesine,Vinorelbina, Vinorelbine, duanomycin, dactinomysin, esorubisin,mafosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, MitomycinC, mithramycin, prednisone, hydroxyprogesterone, testosterone,tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine,amsacrine, chlorambudil, methylcyclohexylnitrosurea, nitrogen mustards,Cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, deoxyco-formycin, 4-hydroxyperoxycyclophosphoramide,5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine,trimetrexate, teni-poside, diethylstilbestrol.

Reference to “cellular toxin” should also be understood to extend to anyother molecule which is perhaps not traditionally regarded as acytotoxic agent but nevertheless falls within the scope of the presentdefinition on the basis that it induces cellular damage, for example DNAdamage, such as nucleophosmin or agents which induce cellular damage aspart of a synergistic process with another agent. Examples includecatalytic antibodies, prodrugs, CHK1/2 inhibitor (such as CBP-501 orAZD7762), histone deacetylase inhibitor (such as vorinostat), tumournecrosis factor related apoptosis inducing ligand or BH3 mimetic (suchas ABT737), small molecule inhibitors such as the tyrosine kinaseinhibitors imatinib mesylate (Glivec®), gefitinib (Iressa®) anderlotinib (Tarceva®), and the monoclonal antibodies (mAb) such asrituximab (Mabthera®) and trastuzumab (Herceptin®).

In yet another embodiment, combination treatments may include, forexample, gemcitabine together with a CHK1/2 inhibitor or irinotecamtogether with a CHK1/2 inhibitor.

The particulate material and/or the stabiliser may be coupled to aligand to effect more specific targeting to a tumour. This will notnecessarily be applicable in every situation but, to the extent that anappropriate target molecule exists for a given tumour, this may provideadditional useful specificity.

According to such an embodiment, there is provided a method of treatinga solid tumour in a subject, said method comprising co-administering tosaid subject an effective amount of particulate material and a cellulartoxin for a time and under conditions sufficient to facilitatedistribution of said particulate material and toxin to said tumour,wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a stabiliser; and    -   (ii) said stabiliser comprising an anchoring portion that (a)        anchors the stabiliser to the particulate material, and (b) is        different from the remainder of the stabiliser;        wherein the particulate material and/or the stabiliser is        linked, bound or otherwise associated with a ligand directed to        a tumour molecule and wherein said particulate material and        toxin penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a stericstabilising polymeric segment and an anchoring portion, wherein thesteric stabilising polymeric segment is different from the anchoringportion, there is also provided a method of treating a solid tumour in asubject, said method comprising co-administering to said subject aneffective amount of particulate material and a cellular toxin for a timeand under conditions sufficient to facilitate distribution of saidparticulate material and toxin to said tumour, wherein:

-   -   (i) said particulate material is administered in the form of a        dispersion in a liquid carrier, the particulate material being        maintained in the dispersed state by a steric stabiliser; and    -   (ii) said steric stabiliser comprising a steric stabilising        polymeric segment and an anchoring portion, wherein the steric        stabilising polymeric segment is different from the anchoring        portion, and wherein the anchoring portion anchors the        stabiliser to the particulate material;        wherein the particulate material and/or the steric stabiliser is        linked, bound or otherwise associated with a ligand directed to        a tumour molecule and wherein said particulate material and        toxin penetrate said solid tumour.

In yet another aspect, there is provided the use of particulate materialand a cellular toxin in the manufacture of a medicament for thetreatment of a solid tumour wherein:

-   -   (i) said particulate material is in the form of a dispersion in        a liquid carrier, the particulate material being maintained in        the dispersed state by a stabiliser; and    -   (ii) said stabiliser comprising an anchoring portion that (a)        anchors the stabiliser to the particulate material, and (b) is        different from the remainder of the stabiliser; and wherein said        particulate material and toxin penetrate said solid tumour.

Where the stabiliser is a steric stabiliser comprising a stericstabilising polymeric segment and an anchoring portion, wherein thesteric stabilising polymeric segment is different from the anchoringportion, there is provided the use of particulate material and acellular toxin in the manufacture of a medicament for the treatment of asolid tumour wherein:

-   -   (i) said particulate material is in the form of a dispersion in        a liquid carrier, the particulate material being maintained in        the dispersed state by a steric stabiliser; and    -   (ii) said steric stabiliser comprising a steric stabilising        polymeric segment and an anchoring portion, wherein the steric        stabilising polymeric segment is different from the anchoring        portion, and wherein the anchoring portion anchors the        stabiliser to the particulate material;        and wherein said particulate material and toxin penetrate said        solid tumour.

Further aspects and/or embodiments of the invention are discussed inmore detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sterically stabilised nanoparticles are able to penetrate intospheroids. TEM images of the accumulation of NP2 particles in spheroids.Arrows indicate areas of nanoparticle accumulation. Boxed region isenlarged and shown in the image on right. Scale bars as indicated.

FIG. 2: Nanoparticles can influence the diffusion of fluorescent activecompounds. Co-administration of the fluorescent active compounds a)doxorubicin and b) mitoxantrone with nanoparticles from examples 2, 3,and 5. Single confocal images of fluorescent drug diffusion into DLD-1spheroids. Scale bar 200 μm.

FIG. 3: The majority of nanoparticles tested did not affect cellularoutgrowth from spheroids. Plot of normalised cellular outgrowth asdescribed in example 29 of the nanoparticles listed in examples 1, 2, 4,8, 9, 12, 13, 15, 16, and 18. Error bars represent standard error.

FIG. 4: Composition of the nanoparticle core does not influencenanoparticle effectiveness. Plot of normalised cellular outgrowth asdescribed in example 29 of the nanoparticles from examples 2, 4, 6, 7,9, 10, 11, 12, 13, 14, 16, 17, and 18, co-administered with doxorubicin.The untreated control spheroids had a normalised outgrowth value of 331%+/−23. Error bars represent standard error.

FIG. 5: Nanoparticle size does not correlate with nanoparticleeffectiveness. Plot of normalised cellular outgrowth as described inexample 29 of the nanoparticles listed in examples 1, 2, 4, 7, 9, 10,11, 12, 13, 14, 16, 17, and 18 co-administered with doxorubicin. Theuntreated control spheroids had a normalised outgrowth value of 331%+/−23. Error bars represent standard error.

FIG. 6: Nanoparticles stabilised with 5-10% amine functionalised polymerincrease the effectiveness of doxorubicin. Plot of normalised cellularoutgrowth as described in example 29 of the nanoparticles listed inexamples 2, 3, 4, 5, 20, 21, 22, and 24, co-administered withdoxorubicin. The untreated control spheroids had a normalised outgrowthvalue of 331% +/−23. Error bars represent standard error.

FIG. 7: Effectiveness of co-administration of NPs with 5% aminefunctionalised stabiliser end group coatings with different cores anddoxorubicin compared to doxorubicin alone. Plot of normalised cellularoutgrowth as described in example 29 of the nanoparticles listed inexamples 2, 8, 9, and 12 co-administered with doxorubicin. The untreatedcontrol spheroids had a normalised outgrowth value of 331% +/−23. Errorbars represent standard error.

FIG. 8: The effect of the active compounds when co-administered withnanoparticles on the viability of spheroids made from two differentcancer cell lines. Plot of normalised cellular outgrowth as described inexample 29 of the nanoparticles listed in examples 2, 3, 4, and 5co-administered with active compounds (Table 2). The untreated DLD-1control spheroids had a normalised outgrowth value of 331% +/−23. Theuntreated PA-1 control spheroids had a normalised outgrowth value of294% +/−21. Error bars represent standard error.

FIG. 9: Effect of delayed administration of active compound compared toco-administration of active compound and nanoparticles. Plot ofnormalised cellular outgrowth as described in example 29 of thenanoparticles listed in examples 2, 3, 4, and 5 DLD-1 spheroids wereeither co-administered nanoparticles and active compound (light greybars) or administered nanoparticles, then 24 hours later treated withactive compound (dark grey bars). The untreated DLD-1 control spheroidshad a normalised outgrowth value of 331% +/−23. Error bars representstandard error.

FIG. 10: Effect of delayed administration of active compound compared toco-administration of active compound and nanoparticles. Plot ofnormalised cellular outgrowth as described in example 29 of thenanoparticles listed in examples 2, 3, 4, and 5. PA-1 spheroids wereeither co-administered nanoparticles and active compound (light greybars) or administered nanoparticles, then 24 hours later treated withactive compound (dark grey bars).The untreated PA-1 control spheroidshad a normalised outgrowth value of 294% +/−21. Error bars representstandard error.

FIG. 11: The most effective co-administered nanoparticle and activecombinations for DLD-1 and PA-1 cells. Plot of normalised cellularoutgrowth as described in example 29 of the nanoparticles listed inexamples 2, 5, 14, 20, 21, and 22, co-administered with active compoundsin DLD-1 spheroids (A) and PA-1 spheroids (B). Error bars representstandard error.

FIG. 12: The co-administration of NP2 but not NP19 or NP23 withdoxorubicin promotes doxorubicin diffusion throughout the spheroid.Confocal images of doxorubicin diffusion in spheroids treated with 1 μMDoxorubicin and the nanoparticles as indicated. Scale bar 200 μm.

FIG. 13 shows a schematic illustration of stabilised particulatematerial that may be used in accordance with the present invention.

FIG. 14 shows a schematic illustration of stabilised particulatematerial that may be used in accordance with the present invention.

FIG. 15 shows a schematic illustration showing the hydrodynamic volumeof a stabilised particulate material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatparticulate material which is maintained in a dispersed state by acertain type of stabiliser can achieve a deeper and more effectivepenetration into a solid tumour than has been previously achievableusing particle technology. The nature of the penetration effected bythese particulate materials has achieved both a significantly widercellular distribution, within the tumour, of the toxin co-administeredwith the particulate material and, further, more effective induction ofcellular toxicity. Since reticuloendothelial clearance from the sites oftumours is significantly less effective than in normal tissue, themethod of the invention enables not only more effective tumourpenetration but, further, the delivery of lower concentrations ofcellular toxins which are enabled to localise, and thereby concentrate,at tumour sites. This reduces the side effects which would be apparentin the context of conventional systemic chemotherapy where suchtreatment would be delivered at the highest dose which can be toleratedby the patient and, further, often in the context of multiple repeatedrounds over a period of months. This development now provides arealistic means of moving away from the treatment of primary tumours andmetastatic disease via the non-targeted, systemic delivery ofchemotherapy.

Reference to a “solid tumour” herein should be understood as a referenceto an encapsulated or unencapsulated mass or other form of growth orcellular aggregate which comprises neoplastic cells. Reference to a“neoplastic cell” should be understood as a reference to a cellexhibiting abnormal growth. The term “growth” should be understood inits broadest sense and includes reference to proliferation. The phrase“abnormal growth” in this context is intended as a reference to cellgrowth which, relative to normal cell growth, exhibits one or more of anincrease in the rate of cell division, an increase in the number of celldivisions, a decrease in the length of the period of cell division, anincrease in the frequency of periods of cell division or uncontrolledproliferation and evasion of apoptosis. Without limiting the presentinvention in any way, the common medical meaning of the term “neoplasia”refers to new cell growth that results as a loss of responsiveness tonormal growth controls, e.g. to neoplastic cell growth. Neoplasiasinclude “tumours” which may be either benign, pre-malignant ormalignant. The term “neoplasm” should be understood as a reference to alesion, tumour or other encapsulated or unencapsulated mass or otherform of growth or cellular aggregate which comprises neoplastic cells.

The term “neoplasm”, in the context of the present invention should beunderstood to include reference to all types of cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues or organs irrespective of histopathologic type or stateof invasiveness.

The term “carcinoma” is recognised by those skilled in the art andrefers to malignancies of epithelial or endocrine tissues includingrespiratory system carcinomas, gastrointestinal system carcinomas,genitourinary system carcinomas, testicular carcinomas, breastcarcinomas, prostate carcinomas, endocrine system carcinomas andmelanomas. Exemplary carcinomas include those forming from tissue of thebreast. The term also includes carcinosarcomas, e.g. which includemalignant tumours composed of carcinomatous and sarcomatous tissues. An“adenocarcinoma” refers to a carcinoma derived from glandular tissue orin which the tumour cells form recognisable glandular structures.

The neoplastic cells comprising the neoplasm may be any cell type,derived from any tissue, such as an epithelial or non-epithelial cell.Examples of neoplasms and neoplastic cells encompassed by the presentinvention include, but are not limited to central nervous systemtumours, retinoblastoma, neuroblastoma and other paediatric tumours,head and neck cancers (e.g. squamous cell cancers), breast and prostatecancers, lung cancer (both small and non-small cell lung cancer), kidneycancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers,hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g.adenocarcinomas and islet cell tumours), colorectal cancer, cervical andanal cancers, uterine and other reproductive tract cancers, urinarytract cancers (e.g. of ureter and bladder), germ cell tumours (e.g.testicular germ cell tumours or ovarian germ cell tumours), ovariancancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary,human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma),lymphomas, malignant melanomas, sarcomas, endocrine tumours (e.g. ofthyroid gland), mesothelioma and other pleural or peritoneal tumours,neuroendocrine tumours and carcinoid tumours.

Preferably, the present invention is directed to the treatment of amalignant neoplastic condition and even more preferably a metastaticneoplastic condition. It would be appreciated that although the methodof the invention can be applied to the treatment of any neoplasm, it isparticularly useful in terms of the treatment of metastasised neoplasms.Without limiting the present invention to any one theory or mode ofaction, non-metastasised primary tumours are treatable either by themethod of the invention or by conventional treatment regimes such assurgical excision of the tumour or radiotherapy. However, tumours whichhave metastasised are not curable by either of these conventionaltreatment regimes due to the often extensive spread and growth ofmetastatic nodules. Accordingly, such conditions are currently onlytreatable by the administration of systemic chemotherapy, this treatmentregime often causing severe side effects for limited curative potential.Still further, even in the context of primary tumours which appear notto have metastasised, chemotherapy is still often recommended followingsurgery and radiation in case metastatic spread has occurred but is notyet detectable. This is a particularly common practice in the context ofcancers which are traditionally regarded as aggressive, such as breastand colon cancers. The method of the present invention now provides analternative to the application of aggressive systemic chemotherapytreatment regimes. Since the systemic administration of the cytotoxicagent of the present invention is able to be delivered in a morelocalised fashion to tumours and is more effectively metabolised by theneoplastic cells, the occurrence of side effects can be minimised viathe administration of lower doses of the cellular toxin.

In one embodiment, said solid tumour is benign.

In another embodiment, said solid tumour is malignant.

Preferably, said malignant solid tumour is a metastatic malignant solidtumour. Reference to “metastatic” should be understood as a reference toa tumour which either has undergone metastatisation or may haveundergone metastatisation.

In one embodiment, said malignant solid tumour is a central nervoussystem tumour, retinoblastoma, neuroblastoma, paediatric tumour, headand neck cancer such as squamous cell cancer, breast and prostatecancer, lung cancer, kidney cancers, such as renal cell adenocarcinoma,oesophagogastric cancer, hepatocellular carcinoma, pancreaticobiliaryneoplasia, such as adenocarcinomas and islet cell tumours, colorectalcancer, cervical cancer, anal cancer, uterine or other reproductivetract cancer, urinary tract cancer, such as of the ureter or bladder,germ cell tumour such as a testicular germ cell tumour or ovarian germcell tumour, ovarian cancer, such as an ovarian epithelial cancer,carcinoma of unknown primary, human immunodeficiency associatedmalignancy, such as Kaposi's sarcoma, lymphoma, leukemia, malignantmelanoma, sarcoma, endocrine tumour, such as of the thyroid gland,mesothelioma or other pleural or peritoneal tumour, neuroendocrinetumour or carcinoid tumour.

As detailed hereinbefore, the method of the present invention is basedon the co-administration of a cellular toxin with stabilised particulatematerial. Previous attempts at using particulate material, such asnanoparticles, to target tumours for either diagnostic or therapeuticpurposes have been extensive but, in the context of therapeutics, ofminimal success. With diagnostics, relatively shallow penetration of theparticles into the tumour has been sufficient to achieve the objectiveof visualising the tumour. However, in terms of the delivery of atherapeutic agent, such shallow penetration has not been sufficient toeffectively deliver the agent throughout the tumour, in particular tothe interior of the tumour. In relation to therapeutics, specifically,conjugation of particles to a wide variety of different materials has sofar failed to live up to the promise of achieving effective tumourpenetration, this being an essential prerequisite for a therapeutic tohave any chance of effectiveness.

Significant effort has also been made to take advantage of the enhancedpermeability and retention (EPR) effect of tumours as a means to developan effective therapeutic. Without limiting the present invention to anyone theory or mode of action, this is a well described phenomenon basedon the notion that certain sizes of molecules, typically liposomes ormacromolecular drugs, tend to preferentially accumulate in tumourtissue. The general explanation for this phenomenon is that, in orderfor tumour cells to grow quickly, they must stimulate the production ofblood vessels. VEGF and other growth factors are involved in cancerangiogenesis. Tumour cell aggregates of sizes as small as 150-200 μmbecome dependent on blood supply carried by neovasculature for theirnutritional and oxygen supply. These newly formed tumour vessels areusually abnormal in form and architecture. They comprise poorly-aligneddefective endothelial cells with wide fenestrations, lacking a smoothmuscle layer, or innervation with a wider lumen, and impaired functionalreceptors for angiotensin II. Furthermore, tumour tissues usually lackeffective lymphatic drainage. All these factors will lead to abnormalmolecular and fluid transport dynamics, especially for macromoleculardrugs. Accordingly, it has been thought that one way to achieveselective drug targeting to solid tumours is to exploit theseabnormalities of tumour vasculature in terms of active and selectivedelivery of anticancer drugs to tumour tissues, notably defining the EPReffect of macromolecular drugs in solid tumours. Due to their largemolecular size, nanosized macromolecular anticancer drugs administeredintravenously escape renal clearance. Often they cannot penetrate thetight endothelial junctions of normal blood vessels, but they canextravasate in tumour vasculature and become trapped in the tumourvicinity. Nevertheless, the EPR effect has not been efficiently orsuccessfully harnessed.

Various nanoparticles have been designed which are directed to achievingefficient cellular endocytosis. However, even if this is achievable, theissue of tissue penetration is still a separate one which, to date, hasnot been successfully overcome. The general notion of the use of ananoparticle as a vector for delivery of a drug is widely discussed inthe literature but, in the absence of achieving deep tumour penetration,is of limited value.

Even where effective tumour distribution of a drug is achieved (bywhatever means) a further problem has been the fact that neoplasticcells within solid tumours can exhibit a slowed metabolism. This meansthat even if a cytotoxic drug penetrates to these cells, if it is noteffectively metabolised it will have a limited impact on the viabilityof the tumour.

Without limiting the present invention to any one theory or mode ofaction, the method of the present invention is thought to achieve itstherapeutic outcomes by both deep penetration of the tumour by theparticulate material, which thereby enables simultaneous or sequentialpenetration by a cellular toxin, and enabling effective metabolism ofthe toxin so as to achieve cell death. Still without limiting thepresent invention in any way, it is thought that this may be due to theparticulate material defined herein, by virtue of their design, actingto upregulate cellular metabolism which has become slowed or dormant.

The cellular toxin of the present invention should be understood as anyproteinaceous or non-proteinaceous molecule or group of molecules whichwill either retard cell growth or induce cell death, for example eitherby directly killing the cell or else delivering a signal which inducesapoptosis. That is, the agent may be either cytostatic or cytocidal. Itwould be appreciated by the person of skill in the art that the methodof the present invention can be designed to deliver one cellular toxinor multiple cellular toxins (i.e. a “cocktail” of drugs). The decisionin relation to how best to proceed can be made by the person of skill inthe art as a matter of routine procedure. For example, depending on thetumour type, certain specific drugs or combinations of drugs areregarded as particularly desirable to use. It would be appreciated thatthe body of knowledge in relation to the characteristics and use ofcytotoxic agents is extensive and the person of skill in the art coulddesign an administration protocol to meet the parameters of the presentinvention as a matter of routine procedure.

Reference to “cellular toxin” herein should therefore be understood as areference to any agent which acts to damage or destroy cells. Withoutlimiting the present invention to any one theory or mode of action, manysuch agents function via the induction of apoptotic processes. However,this is not the only mechanism by which such agents function and it isconceivable that the subject damage or cell death may be induced by someother mechanism. Examples of cytotoxic agents include, but are notlimited to, Actinomycin D, Adriamycin, Arsenic Trioxide, Asparaginase,Bleomycin, Busulfan, Camptosar, Carboplatinum, Carmustine, Chlorambucil,Cisplatin, Corticosteroids, Colicheamicin, Cyclophosphamide,Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Fluorouracil, Gemcitabina, Gemcitabine, Gemzar,Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Melphalan,Mercaptomurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin,Paclitaxel, Platinol, Platinex, Procarbizine, Raltitrexeel, Rixin,Steroids, Streptozocin, Taxol, Taxotere, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulfan, Trihydrate, Vinblastine, Vincristine, Vindesine,Vinorelbina, Vinorelbine, duanomycin, dactinomysin, esorubisin,mafosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, MitomycinC, mithramycin, prednisone, hydroxyprogesterone, testosterone,tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine,amsacrine, chlorambudil, methylcyclohexylnitrosurea, nitrogen mustards,Cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, deoxyco-formycin, 4-hydroxyperoxycyclophosphoramide,5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine,trimetrexate, teni-poside, diethylstilbestrol.

However, reference to “cellular toxin” should also be understood toextend to any other molecule which is perhaps not traditionally regardedas a cytotoxic agent but nevertheless falls within the scope of thepresent definition on the basis that it induces cellular damage, forexample DNA damage, such as nucleophosmin or agents which inducecellular damage as part of a synergistic process with another agent.Examples include catalytic antibodies, prodrugs, CHK1/2 inhibitor (suchas CBP-501 or AZD7762), histone deacetylase inhibitor (such asvorinostat), tumour necrosis factor related apoptosis inducing ligand orBH3 mimetic (such as ABT737), small molecule inhibitors such as thetyrosine kinase inhibitors imatinib mesylate (Glivec®), gefitinib(Iressa®) and erlotinib (Tarceva®), and the monoclonal antibodies (mAb)such as rituximab (Mabthera®) and trastuzumab (Herceptin®).

In yet another embodiment, combination treatments may include, forexample, gemcitabine together with a CHK1/2 inhibitor or irinotecamtogether with a CHK1/2 inhibitor.

In a still further embodiment, the cellular toxin may be a moleculewhich functions as an RNA interference mechanism. Without limiting thepresent invention to any one theory or mode of action “RNA interference”broadly describes a mechanism of gene silencing which is based ondegrading or otherwise preventing the translation of mRNA in a sequencespecific manner. In terms of the application of this technology toselectively knocking down gene expression, exogenous double stranded RNA(dsRNA) specific to a gene sought to be knocked down can be introducedinto the intracellular environment. Once the dsRNA enters the cell, itis cleaved by an RNaseIII-like enzyme, Dicer, into double stranded smallinterfering RNAs (siRNAs) 21-23 nucleotides in length that contain 2nucleotide overhangs on the 3′ ends. In an ATP dependent step, thesiRNAs become integrated into a multi-subunit protein complex known asthe RNAi induced silencing complex (RISC), which guides the siRNAs tothe target RNA sequence. The siRNA unwinds and the antisense strandremains bound to RISC and directs degradation of the complementarytarget mRNA sequence by a combination of endo- and exonucleases.However, whereas the RNAi mechanism was originally identified in thecontext of its role as a microbial defence mechanism in highereukaryotes, it is also known that RNAi based gene expression knockdowncan also function as a mechanism to regulate endogenous gene expression.Specifically, microRNA (miRNA) is a form of endogenous single-strandedRNA which is typically 20-25 nucleotides and is endogenously transcribedfrom DNA, but not translated into protein. The DNA sequence that codesfor an miRNA gene generally includes the miRNA sequence and anapproximate reverse complement. When this DNA sequence is transcribedinto a single-stranded RNA molecule, the miRNA sequence and itsreverse-complement base pair to form a double stranded RNA hairpin loop,this forming the primary miRNA structure (pri-miRNA). A nuclear enzymecleaves the base of the hairpin to form pre-miRNA. The pre-miRNAmolecule is then actively transported out of the nucleus into thecytoplasm where the Dicer enzyme cuts 20-25 nucleotides from the base ofthe hairpin to release the mature miRNA.

Although both of the RNA interference mechanisms detailed aboveeffectively achieve the same outcome, being selective gene expressionknockdown, RNAi based on the use of exogenously administered dsRNAgenerally results in mRNA degradation while RNAi based on the actions ofmiRNAs generally results in translational repression by a mechanismwhich does not involve mRNA degradation. The RNA interference which iscontemplated in the context of the present invention should beunderstood to encompass reference to both of these RNAi gene knockdownmechanisms. The induction of this miRNA based knockdown mechanism couldbe achieved by administering, in accordance with the method of theinvention, exogenous RNA oligonucleotides of the same sequence as anmiRNA, pre-miRNA or pri-miRNA molecules. However, it should beunderstood that these exogenous RNA oligonucleotides may lead to eithermRNA degradation (analogous to that observed with the introduction of anexogenous siRNA population) or mRNA translateral repression, this beingakin to the mechanism by which the endogenous miRNA molecules function.In terms of the objective of the present invention, the occurrence ofeither gene knockdown mechanism is acceptable.

The RNA interference mechanism herein discussed is effected via the useof an RNA oligonucleotide which can induce an RNA interferencemechanism. Reference to an “RNA oligonucleotide” should therefore beunderstood as a reference to an RNA nucleic acid molecule which isdouble stranded or single stranded and is capable of either effectingthe induction of an RNA interference mechanism directed to knocking downthe expression of a gene targeted or downregulating or preventing theonset of such a mechanism. In this regard, the subject oligonucleotidemay be capable of directly modulating an RNA interference mechanism orit may require further processing, such as is characteristic of hairpindouble stranded RNA which requires excision of the hairpin region,longer double stranded RNA molecules which require cleavage by dicer orprecursor molecules such as pre-miRNA which similarly require cleavage.The subject oligonucleotide may be double stranded (as is typical in thecontext of effecting RNA interference) or single stranded (as may be thecase if one is seeking only to produce a RNA oligonucleotide suitablefor binding to an endogenously expressed gene). Examples of RNAoligonucleotides suitable for use in the context of the presentinvention include, but are not limited to:

-   -   (i) long double stranded RNA (dsRNA)—these are generally        produced as a result of the hybridisation of a sense RNA strand        and an antisense RNA strand which are each separately        transcribed by their own vector. Such double stranded molecules        are not characterised by a hairpin loop. These molecules are        required to be cleaved by an enzyme such as Dicer in order to        generate short interfering RNA (siRNA) duplexes. This cleavage        event preferably occurs in the cell in which the dsRNA is        transcribed.    -   (ii) hairpin double stranded RNA (hairpin dsRNA)—these molecules        exhibit a stem-loop configuration and are generally the result        of the transcription of a construct with inverted repeat        sequences which are separated by a nucleotide spacer region,        such as an intron. These molecules are generally of longer RNA        molecules which require both the hairpin loop to be cleaved off        and the resultant linear double stranded molecules to be cleaved        by Dicer in order to generate siRNA. This type of molecule has        the advantage of being expressible by a single vector.    -   (iii) short interfering RNA (siRNA)—these can be synthetically        generated or, recombinantly expressed by the promoter based        expression of a vector comprising tandem sense and antisense        strands each characterised by its own promoter and a 4-5        thymidine transcription termination site. This enables the        generation of 2 separate transcripts which subsequently anneal.        These transcripts are generally of the order of 20-25        nucleotides in length. Accordingly, these molecules require no        further cleavage to enable their functionality in the RNAi        pathway.    -   (iv) short hairpin RNA (shRNA)—these molecules are also known as        “small hairpin RNA” and are similar in length to the siRNA        molecules but with the exception that they are expressed from a        vector comprising inverted repeat sequences of the 20-25        nucleotide RNA molecule, the inverted repeats being separated by        a nucleotide spacer. Subsequently to cleavage of the hairpin        (loop) region, there is generated a functional siRNA molecule.    -   (v) micro RNA/small temporal RNA (miRNA/stRNA)—miRNA and stRNA        are generally understood to represent naturally occurring        endogenously expressed molecules. Accordingly, although the        design and administration of a molecule intended to mimic the        activity of a miRNA will take the form of a synthetically        generated or recombinantly expressed siRNA molecule, the method        of the present invention nevertheless extends to the design and        expression of oligonucleotides intended to mimic miRNA,        pri-miRNA or pre-miRNA molecules by virtue of exhibiting        essentially identical RNA sequences and overall structure. Such        recombinantly generated molecules may be referred to as either        miRNAs or siRNAs.    -   (vi) miRNAs which mediate spatial development (sdRNAs), the        stress response (srRNAs) or cell cycle (ccRNAs).    -   (vii) RNA oligonucleotides designed to hybridise and prevent the        functioning of endogenously expressed miRNA or stRNA or        exogenously introduced siRNA. It would be appreciated that these        molecules are not designed to invoke the RNA interference        mechanism but, rather, prevent the upregulation of this pathway        by the miRNA and/or siRNA molecules which are present in the        intracellular environment. In terms of their effect on the miRNA        to which they hybridise, this is reflective of more classical        antisense inhibition.

It will be appreciated that the person of skill in the art can determinethe most suitable RNA oligonucleotide for use in any given situation.For example, although it is preferable that the subject oligonucleotideexhibits 100% complementarity to its target nucleic acid molecule, theoligonucleotide may nevertheless exhibit some degree of mismatch to theextent that hybridisation sufficient to induce an RNA interferenceresponse in a sequence specific manner is enabled. Accordingly, it ispreferred that the oligonucleotide of the present invention comprises atleast 70% sequence complementarity, more preferably at least 90%complementarity and even more preferably, 95%, 96%, 97%, 98% 99% or 100%sequence complementarity.

In another example pertaining to the design of oligonucleotides suitablefor use in the present invention, it is within the skill of the personof skill in the art to determine the particular structure and length ofthe subject oligonucleotide, for example whether it takes the form ofdsRNA, hairpin dsRNA, siRNA, shRNA, miRNA, pre-miRNA, pri-miRNA etc. Forexample, it is generally understood that stem-loop RNA structures, suchas hairpin dsRNA and shRNA, are more efficient in terms of achievinggene knockdown than, for example, double stranded DNA which is generatedutilising two constructs separately coding the sense and antisense RNAstrands. Still further, the nature and length of the intervening spacerregion can impact on the functionality of a given stem-loop RNAmolecule. In yet still another example, the choice of long dsRNA, whichrequires cleavage by an enzyme such as Dicer, or short dsRNA (such assiRNA or shRNA) can be relevant if there is a risk that in the contextof the particular cellular environment an interferon response could begenerated, this being a more significant risk where long dsRNA is usedthan where short dsRNA molecules are utilised. In still yet anotherexample, whether a single stranded or double stranded nucleic acidmolecule is required to be used will also depend on the functionaloutcome which is sought. For example, to the extent that one istargeting an endogenously expressed miRNA with an antisense molecule, itwould generally be appropriate to design a single stranded RNAoligonucleotide suitable for specifically hybridising to the subjectmiRNA. However, to the extent that it is sought to induce RNAinterference, a double stranded siRNA molecule is required. This may bedesigned as a long dsRNA molecule which undergoes further cleavage or ansiRNA. Still further, the present invention is preferably designed toresult in the generation of a final effector RNA oligonucleotide (i.e. asiRNA or miRNA molecule) which is preferably less than 30 nucleotides inlength, more preferably 15-25 nucleotides in length and most preferably19, 20, 21, 22 or 23 nucleotides in length.

Stabilised particulate material in accordance with the invention canadvantageously be maintained in a dispersed state at low concentrations.The ability for the particulate material to remain in a dispersed statein a diverse array of liquid carriers (including body fluids) atrelatively low concentration, coupled with the ability to tailor thedesign of the stabiliser on a molecular level (e.g. its composition andmolecular weight), may, without wishing to be limited by theory, play arole in enabling the particulate material to achieve deep penetration ofsolid tumours.

As used herein, the expression “particulate material” is intended toembrace material that is capable of being dispersed throughout theliquid carrier and that presents a surface to which the stabiliser maybe associated.

The particulate material will generally be of a size that is less thanabout 500 nm, less than about 350 nm, less than about 250 nm, less thanabout 100 nm, less than about 50 nm, less than about 25 nm or less thanabout 15 nm.

In one embodiment, said particulate material is about: 2, 4, 6, 8, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 nm.

By having an ability to be dispersed throughout the liquid carrier, itwill be appreciated that the particulate material will be sufficientlyinsoluble in the liquid carrier so as to enable the dispersion to haveeffective application.

The particulate material may be in the form of primary particles, or inthe form or an aggregation of primary particles.

For avoidance of any doubt, reference herein to the “size” of theparticulate material is intended to denote an average size (at leastabout 50 number %) of the particles based on the largest dimension of agiven particle. The size of the particulate material per se isdetermined herein by Transmission Electron Microscopy (TEM).

For avoidance of any doubt, when the particulate material is in the formof an aggregation of primary particles, reference to the size of suchmaterial is intended to be a reference to the largest dimension of theaggregate not the primary particles that form the aggregate.

Apart from having medicinal utility in the context of the presentapplication, there is no particular limitation on composition of theparticulate material. The particulate material may have an organiccomposition or an inorganic composition or a combination thereof. Theparticulate material may be inorganic, organic or a combination thereof.

Examples of particulate material include one or more of a metal, a metalalloy, a metal salt, a metal complex, a metal oxide, an inorganic oxide,a radioactive isotope, a polymer particle, and/or combinations thereof.

More specific examples of particulate materials include gold, silver,boron, and salts, complexes or oxides thereof, calcium carbonate, bariumsulphate, iron oxide, chromium oxide, cobalt oxide, manganese oxide,silicon oxide, iron oxyhydroxide, chromium oxyhydroxide, cobaltoxyhydroxide, manganese oxyhydroxide, chromium dioxide, other transitionmetal oxides, polymers such as polystyrene, poly(methyl methacrylate)and poly(butadiene).

In some embodiments of the invention, it is preferred that theparticulate material is magnetic. Magnetic particulate material that maybe used in accordance with the invention will generally be of a size ofless than about 350 nm. Those skilled in the art will appreciate thatthe composition and/or size of the particles can influence theirmagnetic properties. The magnetic particulate material will generallyexhibit ferromagnetic, ferrimagnetic or superparamagnetic properties.

The specific size of the magnetic particulate material used willgenerally be dictated by the intended application of the compositions.For some applications, it may be desirable for the magnetic particulatematerial to be of a size of less than about 300 nm, for example lessthan about 100 nm, or less than about 50 nm.

There is no particular limitation on the type of magnetic particulatematerial that may be used in accordance with the invention. Examples ofsuitable magnetic materials include, but are not limited to, iron,nickel, chromium, cobalt, oxides thereof or mixtures of any of these.Preferred iron oxide magnetic particulate materials include y-ion oxide(i.e. γ-Fe₂O₃, also known as maghemite) and magnetite (Fe₃O₄).

In some applications, it may be desirable to use magnetic material thatis superparamagnetic (i.e. nano-superparamagnetic particles). As usedherein, the term “superparamagnetic” is intended to mean magneticparticles that do not have the following properties; (i) coercivity,(ii) remanence, or (iii) a hysteresis loop when the rate of change of anapplied magnetic field is quasi static.

The magnetic material is preferably selected from ferrites of generalformula MO.Fe₂O₃ where M is a bivalent metal such as Fe, Co, Ni, Mn, Be,Mg, Ca, Ba, Sr, Cu, Zn, Pt or mixtures thereof, or magnetoplumbite typeoxides of the general formula MO.6Fe₂O₃ where M is a large bivalent ion,metallic iron, cobalt or nickel. Additionally, they could be particlesof pure Fe, Ni, Cr or Co or oxides of these. Alternatively they could bemixtures of any of these.

In one embodiment, the magnetic particulate material is or comprisesiron oxide such as magnetite (Fe₃O₄) or maghemite (γ-Fe₂O₃) with aparticle size preferably less than 50 nm, for example between 2 and 40nm.

Particulate material used in accordance with the invention mayconveniently be prepared using techniques known in the art.

In accordance with the invention, the particulate material is maintainedin the dispersed state by a stabiliser. By being “maintained” in thiscontext is meant that in the absence of the stabiliser the particulatematerial would otherwise flocculate or settle out from the liquidcarrier as sediment. In other words, the stabiliser functions to retainthe particulate material in the dispersed state.

The particulate material is in the form of a dispersion within a liquidcarrier, the particulate material being maintained in the dispersedstate by a stabiliser. By “stabiliser” is meant an agent that associateswith the particulate material and assists with preventing it fromflocculating or otherwise becoming non-dispersed within the liquidcarrier.

The stabiliser used in accordance with the invention comprises ananchoring portion that (a) anchors the stabiliser to the particulatematerial, and (b) is different from the remainder of the stabiliser.

By an “anchoring portion” is meant a moiety such as an atom or group ofcovalently coupled atoms that functions to anchor the stabiliser to theparticulate material.

By an anchoring portion that “anchors” the stabiliser to the particulatematerial is meant it is the anchoring portion per se that directlytethers or binds the stabiliser to the particulate material.

The anchoring portion therefore binds the stabiliser to the particulatematerial.

There is no particular limitation on the way in which the stabiliser isanchored to the particulate material. For example, it may be covalentlycoupled to the particulate material, and/or secured to the particulatematerial through electrostatic forces, hydrogen bonding, ionic charge,van der Waals forces, or any combination thereof.

The stabiliser functions to prevent the particulate material fromflocculating or otherwise becoming non-dispersed (i.e. aggregated)within the liquid carrier through known mechanisms such as stericrepulsion, electrosteric repulsion and/or electrostatic repulsion.

Without wishing to be limited by theory, use of a stabiliser inaccordance with the invention is believed to facilitate (i) transport ofthe particulate material in vivo to the site of the solid tumour, and/or(ii) penetration the particulate material throughout the solid tumour,and/or (iii) uptake by subpopulations of cells within the tumour thatwould not otherwise accumulate effective doses of the cellular toxin.

One or more stabiliser can be used in accordance with the presentinvention. By the anchoring portion being “different” to the remainderof the stabiliser is meant that the anchoring portion has a differentstructure or molecular composition to the rest of the stabiliser. Inother words, the stabiliser will have a stabilising portion (i.e. theportion that functions as a stabilising moiety) and an anchoring portion(i.e. the portion that functions to secure or bind the stabiliser to theparticulate material). The stabilising portion and the anchoring portionare different.

By providing the stabiliser with different structural features that giverise to the stabilising and anchoring functions, it has been found thatpractical effect of both functions can be enhanced. Without wishing tobe limited by theory, a strong association between the particulatematerial and the stabiliser (provided by the anchoring portion), incombination with dedicated stabilising moiety is believed to enable theparticulate material to be maintained in a dispersed state throughout adiverse array of liquid carriers at very low concentrations. Suchproperties make the particulate material well suited to being maintainedin a dispersed state post administration within body fluids.

Those skilled in the art will appreciate that stabilisers with a uniqueanchoring portion can function differently to stabilisers without such aunique anchoring portion. For example, a stabiliser such as polyethyleneglycol (PEG) can adsorb to the surface of a particulate material andfunction as a stabiliser. In that case, any part(s) of the PEG chain,which does not have an anchoring portion that is different from theremainder of the stabiliser, will adsorb in a random fashion giving riseto a non-uniform surface stabilising layer.

In contrast, by providing a stabiliser with a stabilising portion anddifferent anchoring portion arrangement a more controlled and uniformsurface stabilising layer can advantageously be formed. For example thepresence of the unique anchoring portion can promote on the surface ofthe particulate material a brush stabilising layer, where the anchoringportion is secured to the surface of the particulate material and theremainder of the stabiliser (i.e. the stabilising portion) extends outfrom the surface of the particulate material into the liquid carrierakin to the bristles extending from the surface of a brush (hence thename “brush” stabilising layer). As a case in point, the PEG stabilisermentioned above might be functionalised with one or more carboxylic acidgroups at the end of the PEG chain to provide for an anchoring portion.The acid functionalised anchoring portion, which is different from theremainder of the stabiliser, can then secure or bind the PEG chain tothe particulate material and allow it to extend freely into the carrierliquid.

Suitable stabilisers may be nonionic, anionic, cationic, orzwitterionic.

In one embodiment, the particulate material is maintained in thedispersed state by a steric stabiliser, wherein the steric stabilisercomprises a steric stabilising polymeric segment and an anchoringportion, wherein the steric stabilising polymeric segment is differentfrom the anchoring portion, and wherein the anchoring portion binds thestabiliser to the particulate material.

In a similar manner to that outlined above, steric stabilising polymericsegment functions to stabilise the particulate material within theliquid carrier, and the anchoring portion functions to secure thestabiliser to the particulate material. By providing the stabiliser withdifferent structural features that give rise to the steric stabilisingand anchoring functions, it has been found that practical effect of bothfunctions can be enhanced.

Examples of suitable stabilisers include, but are not limited to, thosehaving a polymeric stabilising segment.

The stabilising segment will be soluble in the liquid carrier.

In one embodiment, the polymeric stabilising segment comprises polymerselected from polyacrylamide, polyethylene oxide,polyhydroxyethylacrylate, poly N-isopropylacrylamide,polydimethylaminoethylmethacrylate, polyvinyl pyrrolidone and copolymersthereof.

In another embodiment, the anchoring portion comprises one or morecarboxylic acid groups, one or more phosphate groups, one or morephosphonate groups, one or more phosphinate groups, one or more thiolgroups, one or more thiocarbonylthio groups, one or more sulfonic acidgroups, one or more ethoxysilyl groups, and combinations thereof.

In one embodiment, the anchoring portion is an anchoring polymericsegment and at least one of the steric stabilising and anchoringpolymeric segments comprise polymerised residue of one or moreethylenically unsaturated monomers. Employing at least one suchpolymeric segment is believed to enhance the stabilising properties ofthe steric stabiliser.

In one embodiment, the anchoring portion is an anchoring polymericsegment and at least one of the steric stabilising and anchoringpolymeric segments is derived from one or more ethylenically unsaturatedmonomers that have been polymerised by a living polymerisationtechnique. Employing at least one such polymeric segment is believed toenhance the stabilising properties of the steric stabiliser.

By being a “steric” stabiliser is meant that stabilisation of theparticulate material throughout the liquid carrier occurs as a result ofsteric repulsion forces. Having said this, the steric stabiliser maypresent electrostatic repulsion forces that also assist withstabilisation of the particulate material. The steric stabilisingfunction of the stabiliser used in accordance with the inventiontherefore plays an important role in enabling the particulate materialto be maintained in a dispersed state throughout a diverse array ofliquid carriers, including body fluids.

In one embodiment, the stabiliser used in accordance with the inventioncomprises an ionisable functional group that does not form part of theanchoring portion and presents within the carrier liquid a cationic oranionic charge. Stabiliser comprising such ionisable functional group(s)(e.g. amine or carboxylic acid) may be present in an amount ranging fromabout 2 wt % to about 50 wt %, or about 5 wt % to about 40 wt %,relative to the total wt % of stabiliser used. The use of this type ofstabiliser provides for electrosteric stabilisation. The presence ofsuch stabiliser has surprisingly been found to enhance penetration ofthe particulate material.

In one embodiment, the stabiliser comprises a steric stabilisingpolymeric segment having a terminal (i.e. at the end of the polymersegment or chain) functional group. The functional group may be anionisable functional group, such as one that can provide for a cation(e.g. amine) or an anion (carboxylic acid). In one embodiment theionisable functional group provides for a cation.

In a further embodiment, the stabiliser comprises a steric stabilisingpolymeric segment having a terminal functional group selected from anamine, a carboxylic acid and an alcohol.

The amount of stabiliser used relative to the particulate material willvary depending on the nature of the particulate material, particularlyits size. For example, 1 g of 5 nm particulate material will requiremore stabiliser than 1 g of 1 micron particulate material due to itsincreased surface area. Those skilled in the art will be able todetermine the required amount of stabiliser for a given particulatematerial.

For avoidance of any doubt, reference herein to specific features of the“stabiliser” is intended to embrace all forms of stabiliserscontemplated for use in accordance with the invention (i.e. where thestabiliser comprises an anchoring portion that is different form theremainder of the stabiliser, or where the stabiliser is a stericstabiliser comprising a steric stabilising polymeric segment and ananchoring portion, wherein the steric stabilising polymeric segment isdifferent from the anchoring portion).

In one embodiment, the stabiliser used in accordance with the inventioncomprises a polymeric structure. There is no particular limitation onthe molecular weight of the stabiliser, and this feature of thestabiliser may be dictated in part on the mode by which the dispersionis to be administered to a subject. The stabiliser may, for example,have a number average molecular weight of up to about 160,000, or up toabout 150,000, or up to about 100,000, or up to about 50,000.

In one embodiment, the stabilisers used in accordance with the presentinvention will have a relatively low number average molecular weightcompared with stabilisers conventionally used to stabilise particulatematerial.

In some embodiments of the invention, it may be preferable that thenumber average molecular weight of the stabiliser is less than about30,000, or less than about 20,000, or less than about 10,000, or evenless than about 5,000. The number average molecular weight of thestabiliser may also range from about 1,000 to about 3,000.

Stabilisers used in accordance with the invention having a quite lownumber average molecular weight (e.g. less than about 5,000, preferablyin the range of from about 1,000 to about 3,000) have been found to beparticularly effective at stabilising particulate material in vivo.

Molecular weight values referred to herein are number average molecularweight values (Mn). If appropriate, the molecular weight is to bedetermined using gel permeation chromatography (GPC). GPC can beperformed using polystyrene standards for hydrophobic polymers andpolyethylene oxide standards for hydrophilic polymers.

Those skilled in the art will appreciate that determination of themolecular weight for a block copolymer may require additionalprocedures. For example, it may useful to determine the molecular weightof the first block before the second block is added. If a block is lessthan about 3000 molecular weight this can be determined by electrospraymass spectroscopy (EMS). For higher molecular weigh blocks, GPC can beemployed using polystyrene standards for hydrophobic blocks andpolyethylene oxide standards for hydrophilic blocks.

Determining the molecular weight of an overall block copolymer willtypically depend on the length of the two blocks and their solubilitycharacteristics. The molecular weight of low molecular weight blockcopolymers can be determined by EMS as mentioned above. For highermolecular weight block copolymers for which suitable solvents andstandards can be found, GPC may be used. For example, if both blocks arehydrophobic and soluble in, for example, tetrahydrofuran (THF), GPC canbe carried out against polystyrene standards; if both blocks arehydrophilic and soluble in, for example, THF, it may be useful to usepolyethylene oxide standards, rather than polystyrene standards.However, it may be that the blocks of a block copolymer are toodissimilar to allow for a common dissolving solvent; polyacrylamide-b-polystyrene is an example of such a block copolymer. Inthat case, it will generally be necessary to prepare and characterisethe first block and then grow the second block and calculate themolecular weight of the second block on the basis of the degree ofconversion of monomer to polymer.

Stabilisers used in accordance with the invention can advantageouslyexhibit highly efficient stabilising properties in that stabilisation ofthe particulate material can be achieved at both low and highconcentrations of the particulate material within a liquid carrier. Thestabilisers can also provide for stable dispersions of the particulatematerial throughout a diverse array of liquid carriers, such as thosehaving a high ionic strength (e.g. 0.15 M NaCl solution, and even ashigh as in a saturated NaCl solution at room temperature), and also overa wide pH range. Such properties make the dispersions particularlysuitable for in vivo applications.

Without wishing to be limited by theory, the highly efficientstabilising properties that can be provided by the stabilisers arebelieved to stem at least in part from stabilisers comprising ananchoring portion that is separate to and different from the stabilisingportion and securely anchors the stabiliser to the particulate material.

By reference the stabiliser being “anchored” to the particulatematerial, or wherein the anchoring portion “anchors” the stabiliser tothe particulate material, is meant that the stabiliser is securelyattached to the particulate material within a liquid carrier and canremain so attached in the absence of free stabiliser in the liquidcarrier, where the liquid carrier has a high ionic strength (e.g.saturated aqueous sodium chloride solution), and/or where the liquidcarrier has a low ionic strength (e.g. pure water).

Anchoring of the stabiliser to the particulate material may be achievedas a result of the anchoring portion being (1) covalently coupled to theparticulate material, and/or (2) secured to the particulate materialthrough electrostatic forces, hydrogen bonding, ionic charge, Van derWaals forces, or any combination thereof.

As a result of the stabilisers being anchored to the particularmaterial, the particulate material can be maintained in a dispersedstate within the liquid carrier despite it being present at low or highconcentration and/or the liquid carrier having low or high ionicstrength.

Accordingly, a dispersion of the particulate material in a liquidcarrier in accordance with the invention can be advantageously stable(i.e. does not flocculate) under conditions that conventionallystabilised particulate material would be unstable (i.e. wouldflocculate).

Those skilled in the art will appreciate that stabilisers that are notcovalently bound to particulate material generally stabilise andmaintain particulate material in a dispersed state by existing in astate of equilibrium of being adsorbed to and desorbed from theparticulate material. Accordingly, where a stabiliser is present at arelatively low concentration in a given liquid carrier, the equilibriumis generally shifted in favour of the stabiliser being desorbed from theparticulate material, which in turn results in flocculation of theparticulate material.

Where anchoring occurs by the stabiliser being covalently coupled to theparticulate material, there can of course be no desorption of thestabiliser. Where anchoring occurs by other means, the stabilisers usedin accordance with the present invention are nevertheless securelyattached to the particulate material and therefore undergo little if nodesorption from the particulate material even when present at lowconcentration within the liquid carrier. In other words, when present atlow concentration within the liquid carrier the equilibrium of adsorbedstabilisers used in accordance with the invention is strongly in favourof the stabiliser being adsorbed to the particulate material, which inturn facilitates the particulate material being maintained in adispersed state.

A convenient test to confirm the anchoring characteristic of stabilisersused in accordance with the invention, which in turn may also reflecttheir ability to maintain the particulate material in the requireddispersed state, can be performed by diluting the steric stabilisedparticulate material to 1% solids using a suitable liquid carrier(typically water), centrifuging this solution so that the solids form aplug, and then removing the supernatant liquid to isolate the solidplug. The solid plug is then combined with a suitable liquid carrier(typically water) without adding more stabiliser so as to again form 1%solids. Sodium chloride is then added to the resulting solution to yield10% by weight sodium chloride. If the particulate material can beredispersed in this final solution and remains dispersed for at least 1hour, the steric stabilisers are regarded as being anchored to theparticulate material.

By “steric stabilising polymeric segment” is meant a segment or regionof the steric stabiliser that is polymeric (i.e. formed by thepolymerisation of at least one type of monomer) and that provides forthe steric stabilising function of the steric stabiliser. Forconvenience, the steric stabilising polymeric segment may hereinafter bereferred to as polymeric segment “A”.

As mentioned, the steric stabilising polymeric segment functions tostabilise the particulate material throughout the liquid carrier byproviding steric repulsion forces.

By being polymeric, it will be appreciated that the steric stabilisingsegment comprises polymerised monomer residues. Thus, the segment willcomprise polymerised monomer residues that give rise to the requiredsteric stabilising properties. The polymerised monomer residues thatmake up the steric stabilising polymeric segment may be the same ordifferent.

The steric stabilising polymeric segment may be substituted with amoiety (e.g. an optional substituent as herein defined), or contain apolymerised monomer residue, that gives rise to electrostaticstabilising properties.

To provide the desired stabilising effect, the stabilising portion willbe soluble in at least the liquid carrier. The solubility of a givenstabilising portion in a given liquid carrier can readily be determinedby simply preparing the stabilising portion in isolation and conductinga suitable solubility test in the chosen liquid carrier.

Similarly, to provide the desired steric stabilising effect, the stericstabilising polymeric segment will be soluble in at least the liquidcarrier. The solubility of a given steric stabilising polymeric segmentin a given liquid carrier can readily be determined by simply preparingthe polymeric segment in isolation and conducting a suitable solubilitytest in the chosen liquid carrier.

The stabiliser as a whole, may or may not be soluble in the givencarrier liquid, but will nonetheless present a stabilising portion thatis soluble.

Those skilled in the art will have an understanding of polymericmaterials that may be employed as the steric stabilising polymericsegment, as to the monomers that may be polymerised to form suchpolymers. For example, suitable polymeric materials include, but are notlimited to, polyacrylamide, polyethylene oxide,polyhydroxyethylacrylate, poly N-isopropylacrylamide,polydimethylaminoethylmethacrylate, polyvinyl pyrrolidone and copolymersthereof. Thus, suitable monomers that may be used to form thestabilising polymeric segment include, but are not limited to,acrylamide, ethylene oxide, hydroxyethylacrylate, N-isopropylacrylamide,dimethylaminoethylmethacrylate, vinyl pyrrolidone and combinationsthereof.

The particular steric stabilising polymeric segment used as part of thesteric stabiliser will of course depend upon the nature of the liquidcarrier. For example, if an aqueous liquid carrier is used, the stericstabilising polymeric segment should be soluble in the aqueous media.Those skilled in the art will be able to select an appropriate stericstabilising polymeric segment for the chosen liquid carrier.

By being able to select a specific steric stabilising polymeric segmentindependent of the anchoring portion, steric stabilisers used inaccordance with the invention can advantageously be tailor designed tosuit a particular liquid carrier and thereby maximise the stabilisingproperties of the steric stabiliser.

There is no particular limitation concerning the polymerisation thattechnique may be used to prepare the steric stabilising polymericsegment. Living polymerisation techniques have been found particularlyuseful in that regard. Those skilled in the art will appreciate that“living polymerisation” is a form of radical addition polymerisationwhereby chain growth propagates with essentially no chain transfer andessentially no termination that give rise to dead polymer chains. By a“dead polymer chain” is meant one that can not undergo further additionof monomers.

In a living polymerisation, typically all polymer chains are initiatedat the start of the polymerisation with minimal new chains beinginitiated in latter stages of the polymerisation. After this initiationprocess, all the polymer chains in effect grow at the same rate.Characteristics and properties of a living polymerisation generallyinclude (i) the molecular weight of the polymer increases withconversion, (ii) there is a narrow distribution of polymer chain lengths(i.e. they are of similar molecular weight), and (iii) additionalmonomers can be added to the polymer chain to create block co-polymerstructures. Thus living polymerisation enables excellent control overmolecular weight, polymer chain architecture and polydispersity of theresulting polymer that can not be achieved with non-livingpolymerisation methods.

Suitable living polymerisation techniques may be selected from ionicpolymerisation and controlled radical polymerisation (CRP). Examples ofCRP include, but are not limited to, iniferter polymerisation, stablefree radical mediated polymerisation (SFRP), atom transfer radicalpolymerisation (ATRP), and reversible addition fragmentation chaintransfer (RAFT) polymerisation.

The steric stabilising polymeric segment may be formed by thepolymerisation of one type of monomer or a combination of two or moredifferent monomers. Accordingly, the steric stabilising polymericsegment may be a homopolymeric segment or a copolymeric segment.

Given that the stabilising polymeric segment forms only part of thesteric stabiliser, rather than defining the steric stabilising polymericsegment in terms of its number average molecular weight, it can insteadbe useful to make reference to the number of polymerised monomeric unitsthat collectively form the segment. Thus, although there is noparticular limitation on the number of such units that collectively formthe steric stabilising polymeric segment, in some embodiments of theinvention it may be desirable that the steric stabiliser has arelatively low number average molecular weight. In that case, it ispreferable that the steric stabilising polymeric segment has less thanabout 100, more preferably less than about 50, most preferably fromabout 10 to about 30 polymerised monomer residue units that make up theoverall segment.

The steric stabilisers used in accordance with the invention alsocomprise an anchoring portion. The function of the anchoring portion hasbeen mentioned. Provided that the stabiliser can be suitably anchored tothe particulate material and is different to the steric stabiliser,there is no particular limitation concerning the form of the anchoringportion.

The anchoring portion will be covalently coupled to the stericstabilising segment. For convenience, the anchoring portion may berepresented as “B”. The steric stabilising polymeric segment and theanchoring portion may be covalently coupled by any suitable means. Forexample, the steric stabiliser may be described as or comprising thestructure A-C-B, where A represents the steric stabilising polymericsegment, B represents the anchoring portion and C represents a couplingmoiety. Alternatively, the steric stabilising polymeric segment and theanchoring portion may be directly covalently coupled and therefore thestabiliser can be simplistically described as or comprising thestructure A-B. In that case, A represents the steric stabilisingpolymeric segment and B represents the anchoring portion.

The specific anchoring portion used will generally be dictated by thenature of the particulate material to which it is to be anchored. Thoseskilled in the art will be able to select an appropriate anchoringportion to bind with the surface of a given particulate material.

When selecting the steric stabilising segment and anchoring portion, itmay be desirable to consider the properties of these respectivecomponents in the context of the intended application of the dispersion.For example, one or both of the steric stabilising segment and anchoringportion may be selected such that they are biodegradable and/orbiocompatible.

The anchoring portion may be present as one or more moieties that form acovalent bond with the particulate material so as to covalently couplethe stabiliser to the particulate material. For example, the anchoringportion (in an anchored state) may be derived from a thiol moiety (—SH)that covalently couples the stabiliser to the particulate material via a—S— linkage. In other words, the stabiliser used comprises a thiolmoiety, but it will be covalently coupled to the particle via a —S—linkage. Accordingly, reference to a stabiliser “used” in accordancewith the invention is intended to be a reference to the form of thestabiliser prior to it being anchored to the particulate material.

The anchoring portion may be a polymeric segment, or in other words ananchoring polymeric segment. In this form, anchoring of the stabiliserto the particulate material will generally not be by way of covalentcoupling but rather by way of electrostatic forces, hydrogen bonding,ionic charge, Van der Waals forces, or any combination thereof.

By an “anchoring polymeric segment” is meant a segment or region of thesteric stabiliser that is polymeric and that has an affinity toward thesurface of the particulate material and functions to anchor or bind thesteric stabiliser to the particulate material. For convenience, theanchoring polymeric segment may also be represented as “B”.

By being polymeric, it will be appreciated that the anchoring segmentcomprises polymerised monomer residues. The segment will comprisepolymerised monomer residues that give rise to the required anchoring tothe particulate material. The polymerised monomer residues that make upthe anchoring polymeric segment may be the same or different.

The anchoring polymeric segment can present multiple sites for bindinginteractions with the particulate material and it is believed that thisproperty enables the stabiliser to be anchored securely to theparticulate material despite not being covalently coupled thereto.

Generally, the anchoring polymeric segment will have at least twopolymerised monomer residues that each provides a site for binding withthe particulate material, preferably at least three, more preferably atleast five, still more preferably at least seven, most preferably atleast ten of such polymerised monomer residues. Not all of thepolymerised monomer residues that make up the anchoring polymericsegment are necessarily required to give rise to a binding interactionwith the particulate material, but it is generally preferred that themajority if not all of the polymerised monomer residues that make up theanchoring polymeric segment do give rise to a binding interaction withthe particulate material.

The anchoring polymeric segment may therefore be described as havingmultiple sites that collectively anchor the stabiliser to theparticulate material. Even where a given binding site only provides arelatively weak interaction with the particulate material, the presenceof multiples of such sites within the segment enables it as a whole tobind securely with the particulate material.

The anchoring polymeric segment can also be substituted with a moiety(e.g. an optional substituent as herein defined) that may or may notgive rise to a binding interaction with the particulate material.

The specific anchoring polymeric segment used will generally be dictatedby the nature of the particulate material to which it is to bind.

When describing the interaction of the anchoring polymeric segment withthe particulate material, it can be convenient to refer to thehydrophilic and hydrophobic character of the segment and the particulatematerial. Thus, in general, suitable binding interactions will occurwhen the segment and the particulate material have similar hydrophilicor hydrophobic character. For example, where the particulate materialhas a relatively hydrophilic surface (e.g. its surface can be wettedwith water), then good binding should be attained using an anchoringpolymeric segment that has hydrophilic character (e.g. in its isolatedform the segment would be soluble in an aqueous medium).

Such an example might be realised where the particulate material is of atype that can form a charge on its surface. In that case, it may bedesirable for the segment to comprise polymerised residues of monomersthat can also form a charge (e.g. residues of an ionisable monomer) soas to promote ionic binding between the segment and the particulatematerial. Promoting the formation of such charged species might befacilitated by adjusting the pH of the liquid carrier in which thestabiliser and particulate material reside.

By the term “ionisable monomer” is meant that the monomer comprises afunctional group which can be ionised in solution to form a cationic oranionic group. Such functional groups will generally be capable of beingionised under acidic or basic conditions through loss or acceptance of aproton. Generally, the functional groups are acid groups or basic groups(i.e. groups that can donate or accept a H atom, respectively). Forexample, a carboxylic acid functional group may form a carboxylate anionunder basic conditions, and an amine functional group may form aquaternary ammonium cation under acidic conditions. The functionalgroups may also be capable of being ionised through an ion exchangeprocess.

Examples of suitable ionisable monomers having acid groups include, butare not limited to, methacrylic acid, acrylic acid, itaconic acid,p-styrene carboxylic acids, p-styrene sulfonic acids, vinyl sulfonicacid, vinyl phosphonic acid, monoacryloxyethyl phosphate,2-(methacryloyloxy) ethyl phosphate, ethacrylic acid,alpha-chloroacrylic acid, crotonic acid, fumaric acid, citraconic acid,mesaconic acid, and maleic acid. Examples of suitable ionisable monomerswhich have basic groups include, but are not limited to, 2-(dimethylamino) ethyl and propyl acrylates and methacrylates, and thecorresponding 3-(diethylamino) ethyl and propyl acrylates andmethacrylates.

Those skilled in the art will be able to select an appropriate anchoringpolymeric segment to bind with the surface of a given particulatematerial.

By being able to select a specific anchoring polymeric segmentindependent of the steric stabilising polymeric segment, the stericstabilisers used in accordance with the invention can advantageously betailor designed to suit a particular particulate material and therebymaximise the anchoring properties of the steric stabiliser. For example,it may be desirable that the anchoring polymeric segment comprisecarboxylic acid, phosphinate, phosphonate and/or phosphate functionalgroups. Where the particulate material to which anchoring segment bindscomprises iron (e.g. magnetic iron oxide particulate material), it maybe desirable for the segment to comprise phosphinate, phosphonate,and/or phosphate functional groups. Such segments will generally beformed using monomers that comprise the phosphorous functional groups.

Those skilled in the art will appreciate the variety of polymericmaterials that may be employed as the anchoring polymeric segment, as tothe monomers that may be polymerised to form such polymers. For example,suitable polymeric materials include, but are not limited to,polyacrylic acid, polymethacrylic acid, polystyrene, polyitaconic acid,poly-p-styrene carboxylic acids, poly-p-styrene sulfonic acids,polyvinyl sulfonic acid, polyvinyl phosphonic acid, polymonoacryloxyethyl phosphate, poly-2-(methylacryloyloxy) ethyl phosphate,polyethacrylic acid, poly-alpha-chloroacrylic acid, polycrotonic acid,polyfumaric acid, polycitraconic acid, polymesaconic acid, polymaleicacid, poly-2-(dimethyl amino) ethyl and propyl acrylates andmethacrylates, the corresponding poly-3-(diethylamino) ethyl and propylacrylates and methacrylates, hydrophobic acrylate and methacrylatepolymers, polydimethylaminoethylmethacrylate, and copolymers thereof.Thus, suitable monomers that may be used to form the anchoring polymericsegment include, but are not limited to, acrylic acid, methacrylic acid,itaconic acid, p-styrene carboxylic acids, p-styrene sulfonic acids,vinyl sulfonic acid, vinyl phosphonic acid, monoacryloxyethyl phosphate,2-(methylacryloyloxy) ethyl phosphate, ethacrylic acid,alpha-chloroacrylic acid, crotonic acid, fumaric acid, citraconic acid,mesaconic acid, maleic acid, 2-(dimethyl amino) ethyl and propylacrylates and methacrylates, the corresponding 3-(diethylamino) ethyland propyl acrylates and methacrylates, styrene, hydrophobic acrylateand methacrylate monomers, dimethylaminoethylmethacrylate, andcombinations thereof.

Living polymerisation techniques such as those herein described havebeen found particularly useful in preparing the anchoring polymericsegment.

Where the anchoring portion is an anchoring polymeric segment, at leastone of the steric stabilising and anchoring polymeric segments may bederived from one or more ethylenically unsaturated monomers that havebeen polymerised by a living polymerisation technique. Where only one ofthe segments is derived in this manner, it will preferably be theanchoring polymeric segment.

The anchoring polymeric segment may be formed by the polymerisation ofone type of monomer or a combination of two or more different monomers.Accordingly, the anchoring polymeric segment may be a homopolymericsegment or a copolymeric segment.

Given that the anchoring polymeric segment may form only part of thesteric stabiliser, rather than defining the anchoring polymeric segmentin terms of its number average molecular weight, it can instead beuseful to make reference to the number of polymerised monomeric unitsthat collectively form the segment. Thus, although there is noparticular limitation on the number of such units that collectively formthe anchoring polymeric segment, in some embodiments of the invention itmay be desirable that the steric stabiliser has a relatively low numberaverage molecular weight. In that case, it is preferable that theanchoring polymeric segment has less than about 100, more preferablyless than about 40, still more preferably less than about 30, even morepreferably from about 5 to about 25, most preferably from about 5 toabout 15 polymerised monomer residue units that make up the overallsegment.

When selecting the steric stabilising and anchoring polymeric segment,or the monomers that may be used to prepare them, it may be desirable toconsider the properties of the respective polymeric segments in thecontext of the intended application of the dispersion. For example, oneor both polymeric segment may be selected such that they arebiodegradable and/or biocompatible.

Provided that the stabiliser functions as herein described there is noparticular limitation on how the stabilising polymeric segment and theanchoring polymeric segment are to be spatially arranged.

The steric stabilising polymeric segment and the anchoring polymericsegment may be coupled to each other by any suitable means to form thesteric stabiliser used in accordance with invention. For example, thesteric stabiliser may be described as or comprising the structure A-C-B,where A represents the steric stabilising polymeric segment, Brepresents the anchoring polymeric segment and C represents a couplingmoiety. Alternatively, the steric stabilising polymeric segment and theanchoring polymeric segment may be directly coupled to each other via acovalent bond and therefore the stabiliser can be simplisticallydescribed as or comprising an A-B block copolymer. In that case, Arepresents the steric stabilising polymeric segment and B represents theanchoring polymeric segment.

It will be appreciated from the description above that each of A and Bcan independently be a homopolymer or a copolymer (e.g. random, block,tapered, etc.). The stabiliser may comprise more than one stericstabilising polymeric segment (A) and more than one anchoring polymericsegment (B). For example, the stabiliser may be described as orcomprising an A-B-A block copolymer. In that case, each A represents thesteric stabilising polymeric segment, which may be the same ordifferent, and B represents the anchoring polymeric segment. Thestabiliser might also be described as or comprising a B-A-B blockcopolymer, where each B represents the anchoring polymeric segment,which may be the same or different, and A represents the stericstabilising polymeric segment that is of sufficient chain length suchthat it forms a “loop” that extends into the liquid carrier and performsits stabilising role.

The stabiliser may also have more complex structures such as star andcomb polymer structures. In that case, the anchoring polymeric segment Bmight represent the main polymer backbone of such structures, withmultiple steric stabilising polymeric segments A being attached thereto.

The interaction of a steric stabiliser used in accordance with theinvention (in the form of an A-B block copolymer structure) withparticulate material in the liquid carrier might be illustrated in thenot to scale simplified schematic shown in FIG. 13.

With reference to FIG. 13, the steric stabiliser represented by an A-Bblock copolymer exhibits an affinity toward the surface of theparticulate material (P) through the anchoring polymeric segment (B).The anchoring polymeric segment (B) therefore secures the stericstabiliser to the particulate material. The anchoring polymeric segment(B) provides multiple sites for binding interactions between the segmentand the particulate material. The steric stabilising polymeric segment(A), which is different to segment (B), is soluble in the liquid carrierand functions to maintain the particulate material dispersed throughoutthe liquid carrier. It will be appreciated that in practice the surfaceof the particulate material will have many steric stabilisers securedthereto, and that these have been omitted from the illustration in FIG.13 for clarity.

A similar illustration to that in FIG. 13 is shown in FIG. 14 where thesteric stabiliser used in accordance with the invention is in the formof an A-B-A block copolymer.

At least one of the steric stabilising and anchoring polymeric segmentsmay be derived from one or more ethylenically unsaturated monomers thathave been polymerised by a living polymerisation technique such as ionicpolymerisation, iniferter polymerisation, SFRP, ATRP, and RAFTpolymerisation. Of these living polymerisation techniques, RAFTpolymerisation is preferred.

The stabiliser used according to the invention may be prepared and thenused to stabilise the particulate material. Alternatively, a moiety maybe anchored to the particulate material and that moiety used tofacilitate polymerisation of monomer so as to grow the stabiliser outfrom the particulate material.

Those skilled in the art will appreciate that the stabiliser selectedfor use in accordance with the invention may depend on the nature of theparticulate material being stabilised and the way in which it is to beadministered to a subject. For example, if the particulate material isto be administered intravenously and is required to remain incirculation for some time, the stabiliser may need to be a stericstabiliser as herein described.

If the particulate material is to be administered orally and needs toremain stable in the high acid conditions of the stomach, the stabilisermay also need to be a steric stabiliser as herein described, for examplea steric stabiliser comprising poly acrylamide.

By the particulate material being “dispersed throughout” a liquidcarrier is meant that the particulate material presents as a dispersedphase throughout the liquid carrier which itself, relative to theparticulate material, presents as a continuous liquid medium or phase.In other words, the composition might be described as comprising asuspension or dispersion of the particulate material throughout theliquid carrier.

As used herein, the term “liquid” in the context of the liquid carrieris intended to mean a vehicle in which the particulate material isdispersed throughout and which is in a liquid state at least at thetemperature of intended use in the methods of the invention. Typically,a liquid carrier will be considered to be in a “liquid” state if, in theabsence of a stabiliser, particulate material dispersed throughout thecarrier can flocculate or settle out from the carrier to form sediment.In other words, if the particulate material can move relatively freelyin the vehicle, then it is considered “liquid”.

The liquid carrier may be made up of one or more different liquids.Suitable pharmacologically acceptable liquid carriers are described inMartin, Remington's Pharmaceutical Sciences, 18^(th) Ed., MackPublishing Co., Easton, Pa., (1990). Generally, the liquid carrier willbe an aqueous liquid carrier. Water or soluble saline solutions andaqueous dextrose and glycerol solutions are preferably employed asliquid carriers, particularly for injectable solutions.

The dispersion may comprise one or more pharmacologically acceptableadditives known to those in the art. For example, the liquid carrier maycomprise one or more additives such as wetting agents, de-foamingagents, surfactants, buffers, electrolytes, and preservatives.

The particular nature of the liquid carrier and any additive therein (ifpresent) will in part depend upon the intended application of thecomposition. Those skilled in the art will be able to select a suitableliquid carrier and additive (if present) for the intended application ofthe dispersion.

It should also be understood that the particulate material and/or(steric) stabiliser may also be coupled to a ligand to effect morespecific targeting to a tumour. This will not necessarily be applicablein every situation but, to the extent that an appropriate targetmolecule exists for a given tumour, this may provide additional usefulspecificity.

Although the general notion of targeted therapy is not new, to date thesuccess of targeted therapy has been limited by virtue of meeting thecriteria which have been required of a potential target molecule, thesebeing:

-   -   (i) cell surface location    -   (ii) high cell surface molecule density    -   (iii) lack of internalisation of the molecule; and    -   (iv) lack of appreciable antigen shedding from the cell surface.

Limitations do exist in terms of the identification of such molecules,in particular antigens which are also ideally tumour-specific. However,to the extent that such targets are known for a given situation, theymay be usefully exploited.

To this end, reference to a “ligand” should be understood as a referenceto any molecule having specificity (not necessarily exclusivespecificity, although this is preferable) and binding affinity for atumour molecule. Examples of ligands include immunointeractivemolecules, peptidomimetic agents, lanthamide metals (which interact withRNA species), enzymatic substrates (which interact with celldeath-related enzymes) and putrescine (which interacts with tissuetransglutaminase). In one embodiment, the ligand is an immunointeractivemolecule. Although a preferred immunointeractive molecule is animmunoglobulin molecule, the present invention extends to otherimmunointeractive molecules such as antibody fragments, single chainantibodies, deimmunized antibodies including humanized antibodies andT-cell associated antigen-binding molecules (TABMs). Most preferably,the immunointeractive molecule is an antibody such as a polyclonal ormonoclonal antibody. It should be understood that the subject ligand maybe linked, bound or otherwise associated to any other proteinaceous ornon-proteinaceous molecule or cell.

The ligand is “directed to” the tumour molecule. It should be understoodthat the ligand may not necessarily exhibit complete exclusivity,although this is preferable. For example, antibodies are known tosometimes crossreact with other antigens. An antigenic determinant orepitope includes that part of the molecule to which an immune responsecan be directed. The antigenic determinant or epitope may be a B-cellepitope or where appropriate a T-cell receptor binding molecule.

The present invention may also be designed such that a ligand isdirected to one or more tumour molecules. Accordingly, the presentinvention may be designed to administer two or more ligands directed todifferent targets, for example as a means of increasing the dose ofcellular toxin which is delivered to a population of neoplastic cells.It also provides a convenient means of simultaneously delivering twodifferent toxins.

Where used, the ligand may be directly bound to the particulate materialor indirectly bound to the particulate material by forming part of thesteric stabiliser. For example, the ligand may be bound to the stericstabiliser.

Those skilled in the art will appreciate that the dispersed particulatematerial used in accordance with the invention will present ahydrodynamic diameter within the liquid carrier. The hydrodynamicdiameter is the distance or size that is derived from the particulatematerial per se and the steric stabilisers associated with theparticulate material. This can be more clearly explained with referenceto FIG. 15 where the particulate material per se (10) is dispersedwithin a carrier liquid (not shown) by (steric) stabilisers (20). Thehydrodynamic diameter (30) of the dispersed particulate material cantherefore be seen to represent the diameter afforded by a combination ofthe particulate material and the (steric) stabilisers. Where thedispersed particulate material does not have a symmetrical shape, thehydrodynamic diameter will be considered to be that of the largesthydrodynamic diameter presented by the dispersed particulate material.

Without wishing to be limited by theory, it is believed that thehydrodynamic diameter of the dispersed particulate material may alsoplay a role in facilitating deep penetration of the particulate materialwithin tumours.

In one embodiment, the hydrodynamic diameter of the dispersedparticulate material is less than about 500 nm, is less than about 350nm, less than about 250 nm, less than about 100 nm, less than about 50nm, less than about 25 nm or less than about 15 nm.

In a further embodiment, the hydrodynamic diameter of the dispersedparticulate material is about: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, or 300 nm.

For avoidance of any doubt, reference herein to the “the hydrodynamicdiameter” of the dispersed particulate material is intended to denote anaverage diameter (at least about 50 number %) of the dispersedparticulate material. The hydrodynamic diameter of dispersed particulatematerial is determined herein by Hydrodynamic Chromatography (HDC,PL-PSDA (Polymer Laboratories)).

Reference herein to a “subject” should be understood to encompasshumans, primates, livestock animals (e.g. sheep, pigs, cattle, horses,donkeys), laboratory test animals (e.g. mice, rabbits, rats, guineapigs), companion animals (e.g. dogs, cats) and captive wild animals(e.g. foxes, kangaroos, deer). Preferably, the mammal is a human.

It should be understood that the term “treatment” does not necessarilyimply that a subject is treated until total recovery. Accordingly,treatment includes reducing the severity of an existing condition,amelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition.

An “effective amount” means an amount necessary at least partly toattain the desired response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of a particularcondition being treated. The amount varies depending upon the health andphysical condition of the individual to be treated, the taxonomic groupof individual to be treated, the degree of protection desired, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.

In a related aspect of the present invention, the subject undergoingtreatment may be any human or animal in need of therapeutic treatment.In this regard, reference herein to “treatment” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a mammal is treated until total recovery. Accordingly, treatmentincludes amelioration of the symptoms of a particular condition orpreventing or otherwise reducing the risk of developing a particularcondition. “Treatment” may also reduce the severity of an existingcondition.

Administration of the particulate material and cellular toxin, in theform of pharmaceutical compositions, may be performed by any convenientmeans. The pharmaceutical composition is contemplated to exhibittherapeutic activity when administered in an amount which depends on theparticular case. The variation depends, for example, on the human oranimal and the particular agent, particulate material and toxin selectedfor use. A broad range of doses may be applicable. Dosage regimes may beadjusted to provide the optimum therapeutic response.

Routes of administration include, but are not limited to,respiratorally, intratracheally, nasopharyngeally, intravenously,intraperitoneally, subcutaneously, intracranially, intradermally,intramuscularly, intraoccularly, intrathecally, intracereberally,intranasally, infusion, orally, rectally, via IV drip patch and implant.The particle may also be administered directly to the tumour.

The compositions in accordance with the invention comprisepharmacologically acceptable particulate material dispersed throughout apharmacologically acceptable liquid carrier. By “pharmacologicallyacceptable” is meant that the particulate material, liquid carrier, orother constituent of the composition (e.g. the steric stabiliser) issuitable for administration to a subject in their own right. In otherwords, administration of the particulate material, liquid carrier orother constituent of the composition to a subject will not result inunacceptable toxicity, including allergenic responses and diseasestates.

As a guide only, a person skilled in the art may consider“pharmacologically acceptable” as an entity approved by a regulatoryagency of a federal or state government or listed in the US Pharmacopeiaor other generally recognised pharmacopeia for use in animals, and moreparticularly humans.

Having said this, those skilled in the art will appreciate that thesuitability of a composition for administration to a subject and whetheror not a given particulate material or liquid carrier would beconsidered pharmacologically acceptable, will to some extent depend uponthe mode of administration selected. Thus, the mode of administrationmay need to be considered when evaluating whether a given composition issuitable for administration to a subject or pharmacologicallyacceptable.

The pharmaceutical forms are preferably suitable for injectable use andinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

In one embodiment, the stabilised particulate material and cellulartoxin may be formulated in a single formulation. In an alternativeembodiment, said stabilised particulate material and cellular toxin areformulated in two separate formulations.

The present invention is further described by reference to the followingnon-limiting examples.

EXAMPLE 1 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion Using poly(monoacryloxyethylphosphate)m-Block-poly(acrylamide)₂₀ Macro Raft Agent Part (a):Preparation of Diluted Aqueous Ferrofluid Stable in Acidic Medium

Magnetite nanoparticles were produced following the method of Massart(Preparation of aqueous magnetic liquids in alkaline and acidic media.IEEE Transactions on Magnetics, 1981. MAG-17(2): p. 1247-1248). In atypical reaction, 80 ml of 1M FeCl₃.6H₂O in 2M HCl and 40 ml of 1MFeCl₂.4H₂O in 2M HCl were mixed in a 2 Litre beaker and the mixturediluted to 1.2 Litre with MQ-water. 250 ml of NH₄OH (28% (w/w)) was thenquickly added to the beaker and the mixture vigorously stirred for 30minutes. Upon adding NH₄OH, the colour of the mixture immediately turnedfrom orange to black suggesting the formation of magnetite. Magnetitewas then oxidized in acidic medium to maghemite by heating at 90° C.with iron nitrate for about an hour. The colour of the suspensionchanged from black to reddish brown. Maghemite particles were thenmagnetically decanted, washed with acetone and finally peptized in wateryielding a stable dispersion (5 wt %). The pH of the dispersion wasabout 1.5-2.

Part (b): Preparation of a poly(monoacryloxyethylphosphate)10-Block-poly(acrylamide)20 Macro-RAFT Agent Uing:2-{[(dodecylsulfanyl)carbonothioyl]sulfanyl} succinic acid

A solution of 2-{[(dodecylsulfanyl)carbonothioyl]sulfanyl} succinic acid(0.81 g, 2.0 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.09 g, 0.3 mmol),acrylamide (2.87 g, 40.3 mmol) in dioxane (15 g) and water (15 g) wasprepared in a 100 mL round bottom flask. This was stirred magneticallyand sparged with nitrogen for 15 minutes. The flask was then heated at70° C. for 4 hrs. At the end of this period, monoacryloxyethyl phosphate(3.98 g, 20 mmol) and 4,4′-azobis(4-cyanovaleric acid) (0.09 g, 0.3mmol) were added to the flask. The mixture was deoxygenated and heatingwas continued at 80° C. for a further 12 hours. The copolymer solutionhad 23.6% solids. The copolymer solution was then diluted with MQ waterto 0.7 wt % and the pH adjusted to 5 using 0.1M NaOH.

Part (c): Preparation of Sterically Stabilized Iron Oxide Nanoparticlesfrom the Aqueous Ferrofluid of Example 1, Part (a) and the Macro-RAFTAgent of Example 1, Part (b)

Nanoparticle dispersion prepared in example 1 part (a) (27 g) wasdiluted with MQ water (200 g) to yield a 2 wt % dispersion of thenanoparticles. The pH of this nanoparticle dispersion was then raised to5 using 0.1 M sodium hydroxide. The 2 wt % dispersion of the iron oxidedispersion was then added to the macro-RAFT copolymer solution fromexample 1, part (b) (100 g). The mixture was stirred vigorously with anoverhead stirrer for 45minutes before the pH was adjusted to pH 7 usingsodium hydroxide solution. The mixture was then left to stir vigorouslyfor another 2 hours at room temperature. The nanoparticle dispersion wasthen dialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. Bigger particles inthe dispersion were removed by ultracentrifugation. The purifiednanoparticle dispersion was then distilled to increase the solidsloading in the aqueous ferrofluid dispersion to about 70 wt %. Theresulting aqueous ferrofluid was found to be stable in 60% ammoniumnitrate solution.

EXAMPLE 2 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion Using 95% poly(monoacryloxyethylphosphate)10-Block-poly(ethylene oxide)17 Macro Raft Agent and 5%poly(monoacryloxyethyl phosphate)10-Block-poly(acrylamide)25 Macro RaftAgent Part (a): Esterification of poly(ethylene glycol) monomethyl etherwith 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic Acid

MethoxyPEG (Mn ˜798) was warmed and stirred to liquefy and homogenizeit, and 19.95 g (25.0 mmol) was then weighed into a 250 mL 3-neckedround bottom flask, and then allowed to solidify.2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid (6.96 g, 29.3mmol) and 4-dimethylaminopyridine (360 mg, 2.9 mmol) were added to theflask, a magnetic stirbar was introduced, and the flask was purged withnitrogen. Dry dichloromethane (75 mL) was added and the mixture wasstirred until the solids had all dissolved. The flask was then cooled inan ice bath and a solution of N,N′-dicyclohexylcarbodiimide (6.03 g,29.3 mmol) in dry dichloromethane (25 mL) was then added dropwise over 1h. The reaction was stirred in the ice-bath for a further 10 min, thenat room temperature for 24 h. The resulting yellow slurry was dilutedwith 1:1 hexane-ether (100 mL) and filtered through a sintered glassfunnel. The filter residue was washed with further small portions of 1:1hexane-ether until it was white, and the combined filtrates wereevaporated to give a cloudy and gritty dull orange oil. The crudeproduct was dissolve in dichloromethane (75 mL) and stirred with solidoxalic acid (4 g) for 1 h, then diluted with hexane (70 mL) and allowedto settle, producing a flocculent white precipitate. The mixture wasfiltered and evaporated, and the crude oil was dissolved in 2:1hexane-dichloromethane (150 mL) and passed through a plug of alumina (40g). Elution with further 2:1 hexane-dichloromethane was continued untilthe eluate was colourless. The combined eluates were dried with sodiumsulphate, filtered, and evaporated to give a clear pale orange oil,24.69 g, 97%.

Part (b): Preparation of a poly(ethyleneoxide)17-Block-poly(monoacryloxyethyl phosphate)10 Macro-Raft AgentBased on the Macro-RAFT of Example 2 Part (a)

A solution of RAFT-PEO from example 2 part (a) (3.60 g, 3.5 mmol),4,4′-azobis(4-cyanovaleric acid) (0.20 g, 0.7 mmol), monoacryloxyethylphosphate (6.89 g, 35 mmol) in dioxane (45 g) and water (22.5 g) wasprepared in a 250 mL round bottom flask. This was stirred magneticallyand sparged with nitrogen for 15 minutes and the reaction was carriedout at 70° C. for 12 hours. The copolymer solution had 15.1% solids. Thecopolymer solution was then diluted with MQ water to 0.7 wt % and the pHadjusted to 5 using 0.1M NaOH.

Part (c): Preparation of a poly(monoacryloxyethylphosphate)10-block-poly(acrylamide)20 Macro-RAFT Agent Using2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid(3.2 g, 13.6 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.19 g, 0.7 mmol),acrylamide (19.27 g, 271.1 mmol) in dioxane (45 g) and water (22.5 g)was prepared in a 250 mL round bottom flask. This was stirredmagnetically and sparged with nitrogen for 15 minutes. The flask wasthen placed in a 70° C. for 4 hrs. The homopolymer solution had 32.0%solids. 15 g of the obtained homopolymer solution, monoacryloxyethylphosphate (4.50 g, 22.9 mmol) and 4,4′-azobis(4-cyanovaleric acid) (0.04g, 0.2 mmol) were added to a 100 mL round bottom flask. The mixture wasdeoxygenated by nitrogen sparging for 15 minute and the flask stirred ina 70° C. oil bath for 12 hours. The copolymer solution, which contained40.4% solids, was then diluted with MQ water to 1.2 wt %. The pH of thediluted copolymer solution was adjusted to 5 with 0.1M NaOH.

Part (d): Preparation of Sterically Stabilized Iron Oxide Nanoparticlesfrom the Aqueous Ferrofluid of Example 1 Part (a) and a 95:5 Blend ofthe Macro-RAFT Agent of Example 2 Part (b) and the Macro-RAFT Agent ofExample 2 Part (c)

A nanoparticle dispersion prepared according to example 1, part (a) wasdiluted with MQ water to yield a 2 wt % dispersion of the nanoparticles.The pH of this prepared nanoparticle dispersion was then raised to 5. Ablend of macro-RAFT which consist of 50 g of a 0.7 wt % solution ofexample 2 part (b) and 50 g of 1.2 wt % solution of example 2 part (c)were mixed together and the pH adjusted to 5 using 0.1M NaOH. The 2 wt %dispersion of iron oxide maintained at the same pH was then added to themacro-RAFT blend. The mixture was vigorously stirred for 2 hours at roomtemperature before the pH was adjusted to 7.0. The mixture was then leftstirring for another 3 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. Bigger particles inthe dispersion were removed by ultracentrifugation. The purifiednanoparticle dispersion was then distilled to increase the solidsloading in the aqueous ferrofluid dispersion to about 70 wt %. Theresulting aqueous ferrofluid was found to be stable in phosphatebuffered saline solution.

Part (e): Modification of Stabilisers for Iron Oxide Particles ofExample 2 Part (d)

Into coated nanoparticles prepared from example 2 part (d) (7.8 g),N-hydroxysuccinimide (NHS, 14.4 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 20 mg) were added,mixed by shaking and allowed to react for 2 hours at room temperature. Asolution of diamine (90 mg of 2,2′-(Ethylenedioxy)bis-(ethylamine) in 1ml of water) was then added to the reaction mixture and allowed to reactfor a further 3.5 hours. The solution was then dialysed against excesswater with numerous changes, to remove free EDAC and the reactionby-products.

EXAMPLE 3 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion Using the Ferrofluid of Example 1 Part (a) and thepoly(monoacryloxyethyl phosphate)₁₀-Block-poly(ethylene oxide)₁₇ MacroRaft Agent of Example 2 Part (b)

Nanoparticle dispersion (8.0 g) prepared according to example 1 part (a)was diluted with 50 g of MQ water to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this prepared nanoparticle dispersion was thenraised to 5. The 0.5 wt % dispersion of iron oxide maintained at thesame pH was then added to the 50 g of macro-RAFT agent from example 2part (b). The mixture was vigorously stirred for 2 hours at roomtemperature before the pH was adjusted to 7.0. The mixture was then leftstirring for another 3 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The final solids ofthe dispersion was 0.74%.

EXAMPLE 4 Steric Stabilization of Iron Oxide Nanoparticles of theAqueous Ferrofluid of Example 1 Part (a) Using thepoly(monoacryloxyethyl phosphate)₁₀-block-poly(acrylamide)₂₀ Macro-RaftAgent of Example 2 Part (c)

Nanoparticle dispersion prepared in example 1 part (a) (6.19 g) wasdiluted with MQ water (100 g) to yield a 1 wt % dispersion of thenanoparticles. The pH of this nanoparticle dispersion was then raised to5 using 0.1 M sodium hydroxide. The 2 wt % dispersion of the iron oxidedispersion was then added to the macro-RAFT copolymer solution fromexample 2 part (c) (50 g). The mixture was stirred vigorously with anoverhead stirrer 2 hours before the pH was adjusted to pH 7 using sodiumhydroxide solution. The mixture was then left to stir vigorously foranother 12 hours at room temperature. The nanoparticle dispersion wasthen dialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solid content ofthe final dispersion is 0.71%.

EXAMPLE 5 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion from the Aqueous Ferrofluid of Example 1 Part (a) Using 100%amine Modified poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ Part (a): Preparation of amineModified poly(monoacryloxyethyl phosphate)10-Block-poly(acrylamide)20Macro-RAFT Agent from the Macro-RAFT Agent of Example 2 Part (c)

N-hydroxy succinimide 98% (0.64 g), 2,2′-(Ethylenedioxy)bis-(ethylamine), 98% (0.54 g) was added to 30.0 g ofpoly(monoacryloxyethyl phosphate)₁₀-block-poly(acrylamide)₂₀ blockcopolymer of example 2 part (c) at pH 6.25 in a 100 mL glass bottle. Thebottle was sealed with parafilm and placed on a roller for mixing for 2hours. After 2 hours, the mixture had a pH of 8.12 to yield theNHS-activated carboxyl groups which are reactive towards primary amine.1.26 g of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloridewas then added to the mixture, which was left on the roller for further12 hours. The excess N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride was removed by dialysis.

Part (b): Preparation of Sterically Stabilized Iron Oxide Nanoparticlesfrom the Aqueous Ferrofluid of Example 1, Part (a) and the Macro-RAFTAgent of Example 5, Part (a)

Nanoparticle dispersion prepared in example 1 part (a) (8.38 g) wasdiluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this nanoparticle dispersion was then raised to5 using 0.1 M sodium hydroxide. The 0.5 wt % dispersion of the ironoxide nanoparticles was then added to the macro-RAFT copolymer solution,from example 5 part (a) (22.6 g) and 50 g of MQ water. The mixture wasstirred vigorously with an overhead stirrer for 2 hours before the pHwas adjusted to pH 7 using sodium hydroxide solution. The mixture wasthen left to stir vigorously for another 10 hours at room temperature.The nanoparticle dispersion was then dialysed to remove salts, residualsolvents, unwanted low molecular weight reaction side products andunbound polymer. The solids content of the dialysed aqueous ferrofluiddispersion was 0.36%.

EXAMPLE 6 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion Using 95% poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ Macro-Raft Agent of Example 2 Part(c) and 5% amine Modified poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ of Example 5 Part (a)

Nanoparticle dispersion prepared in example 1 part (a) (8.09 g) wasdiluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this nanoparticle dispersion was then raised to5 using 0.1 M sodium hydroxide. The 0.5 wt % dispersion of the ironoxide dispersion was then added to the macro-RAFT copolymer solutionwhich was pH 5.0 from example 5 part (a) (1.7 g), example 2 part (c)(1.0 g) and 50 g of MQ water. The mixture was stirred vigorously with anoverhead stirrer for 2 hours before the pH was adjusted to pH 7 usingsodium hydroxide solution. The mixture was then left to stir vigorouslyfor another 3 hours at room temperature. The nanoparticle dispersion wasthen dialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solids content ofthe dialysed aqueous ferrofluid dispersion is 0.53%.

EXAMPLE 7 Steric Stabilization of Iron Oxide Nanoparticles in AqueousDispersion Using poly(monoacryloxyethyl phosphate)₁₀-Block-poly(acrylamide)₆₀ Macro Raft Agent Part (a): Preparation ofDiluted Aqueous Ferrofluid Stable in Acidic Medium

Magnetite nanoparticles were produced following the method of Massart(Preparation of aqueous magnetic liquids in alkaline and acidic media.IEEE Transactions on Magnetics, 1981. MAG-17(2): p. 1247-1248). Anaqueous mixture of ferric and ferrous chlorides was added to ammoniasolution. The resulting precipitate was isolated by centrifugation thenoxidised to maghemite by mixing with iron nitrate solution and heating.The precipitate was then washed in 2 molar nitric acid then finallypeptised by water to form a dilute aqueous ferrofluid of approximately 5wt % solids.

Part (b): Preparation of a poly(monoacryloxyethylphosphate)10-Block-poly(acrylamide)60 Macro-RAFT Agent Using2-{[(butylsulfanyl)carbonothioyl]sulfanyl} propanoic acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl} propanoic acid(0.26 g, 1.1 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.06 g, 0.2 mmol),acrylamide (4.73 g, 66 mmol) in dioxane (10 g) and water (10 g) wasprepared in a 100 mL round bottom flask. This was stirred magneticallyand sparged with nitrogen for 15 minutes. The flask was then heated at70° C. for 4 hrs. At the end of this period, monoacryloxyethyl phosphate(2.17 g, 11.1 mmol) and 4,4′-azobis(4-cyanovaleric acid) (0.06 g, 0.2mmol) were added to the flask. The mixture was deoxygenated and heatingwas continued at 80° C. for a further 12 hours. The copolymer solutionhad 24% solids. The copolymer solution was then diluted with MQ water to0.7 wt % and the pH adjusted to 5 using 0.1M NaOH.

Part (c): Preparation of Sterically Stabilized Iron Oxide Nanoparticlesfrom the Aqueous Ferrofluid of Part (a) and the Macro-RAFT Agent ofExample 7 Part (b)

Nanoparticle dispersion prepared in example 7 part (a) (40 g) wasdiluted with MQ water (200 g) to yield a 1 wt % dispersion of thenanoparticles. The pH of this nanoparticle dispersion was then raised to5 using 0.1 M sodium hydroxide. The lwt % dispersion of the iron oxidedispersion was then added to the macro-RAFT copolymer solution from part(b) (200 g). The mixture was stirred vigorously with an overhead stirrerfor 45minutes before the pH was adjusted to pH 7 using sodium hydroxidesolution. The mixture was then left to stirr vigorously for another 2hours at room temperature. The nanoparticle dispersion was then dialysedto remove salts, residual solvents, unwanted low molecular weightreaction side products and unbound polymer. Bigger particles in thedispersion were removed by ultracentrifugation. The purifiednanoparticle dispersion was then distilled to increase the solidsloading in the aqueous ferrofluid dispersion to about 70 wt %.

EXAMPLE 8 Steric Stabilization of Sigma Ludox as 30 Silica ParticlesUsing 95% poly[2-(dimethylamino)ethylmethacrylate]₁₀-Block-poly(ethylene oxide)₁₇ Macro Raft Agent and 5%poly(2-(dimethylamino)ethyl methacrylate)₁₀-Block-poly(acrylamide)25Macro Raft Agent Part (a): Preparation of a poly[2-(dimethylamino)ethylmethacrylate]10-Block-poly(ethylene oxide)17 Macro-RAFT Agent Based on2-{[butylsulfanyl)carbonothioyl]-sulfanyl}propanoic acid

A solution of RAFT-PEO from example 2 part (a) (1.38 g, 1.4 mmol),4,4′-azobis(4-cyanovaleric acid) (0.08 g, 0.3 mmol),2-(Dimethylamino)ethyl methacrylate (2.13 g, 13.6 mmol) in dioxane (10g) and water (5 g) was prepared in a 100 mL round bottom flask. This wasstirred magnetically and sparged with nitrogen for 15 minutes and thereaction was carried out at 70° C. for a 12 hours. The copolymersolution had 18.4% solids.

Part (b): Selective Quaternization of a poly[2-(dimethylamino)ethylmethacrylate]10-Block-poly(ethylene oxide)17 Macro-RAFT Agent Based on2-{[butylsulfanyl)carbonothioyl]-sulfanyl}propanoic Acid

Example 8 part (a), 16.5 g was diluted with MQ water (17 g) and methyliodide (0.7 g) added. The mixture was stirred at room temperature for 1hour before being partially dried using a rotary evaporator. The driedsamples were then placed in the vacuum oven to dry the macro raft agentwhich yielded 100% solids.

Part (c): Preparation of a poly(2-(Dimethylamino)ethylmethacrylate)10-Block-poly(acrylamide)25 Macro-RAFT Agent Based on2-{[butylsulfanyl)carbonothioyl]-sulfanyl}propanoic Acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid(0.6 g, 2.6 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.11 g, 0.4 mmol),acrylamide (4.45 g, 62.7 mmol) in dioxane (18.8 g) and water (10.5 g)was prepared in a 100 mL round bottom flask. This was stirredmagnetically and sparged with nitrogen for 15 minutes. The flask wasthen placed in a 70° C. oil bath for 4 hrs. The homopolymer solution had32.7% solids. All of the homopolymer solution obtained,2-(Dimethylamino)ethyl methacrylate (3.94 g, 25.1 mmol) and4,4′-azobis(4-cyanovaleric acid) (0.088 g, 0.32 mmol) were added to a100 mL round bottom flask. The mixture was deoxygenated for 15 minuteand placed in a 70° C. oil bath for 12 hours. The final solids ofcopolymer solution was 26.9%.

Part (d): Selective Quaternization of a poly(2-(dimethylamino)ethylmethacrylate)10-Block-poly(acrylamide)25 Macro-RAFT Agent Based on2-{[butylsulfanyl)carbonothioyl]-sulfanyl}propanoic Acid

Example 8 part (c) (19.3 g) was diluted with MQ water (20 g) and methyliodide (0.78 g) was added. The mixture was stirred at room temperaturefor 1 hour be partially dried using a rotary evaporator. The partiallydried samples was then placed in the vacuum oven to dry the macro raftagent which yield 100% solids.

Part (e): Preparation of Sterically Stabilized Sigma Ludox AS30 SilicaParticles Using a 95:5 Blend of the Macro-RAFT Agents of Example 8 Part(b) and Example 8 Part (d)

Ludox AS30 from Sigma Aldrich (2.5 g) was diluted with MQ water (100 g)to yield a 2 wt % dispersion of the nanoparticles and the pH is 9.62. Amixture of example 8 part (b) (0.96 g) and of example 8 part (d) (0.0653g) was dissolved in MQ water (50 g) and the pH was 7.59. The 2 wt %dispersion was then poured into the mixture of macro-Raft agents. Themixture was vigorously stirred for 5 hours at room temperature. Thedispersion was then dialysed to remove salts, residual solvents,unwanted low molecular weight reaction side products and unboundpolymer. The solid content of the dialysed silica sol dispersion was0.69%. The pH of the sample was adjusted to 6.76 with sodium hydroxidesolution.

Part (f): Modification of Stabilisers for Silica Particles of Example 8Part (e) [EP341070A]

Sterically stabilised silica sol particles prepared from example 8 part(e) (60 g), N-hydroxysuccinimide (NHS, 39.4 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 56.2 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. 37 mg of 2,2′-(Ethylenedioxy)bis-(ethylamine) was thenadded to the reaction mixture and allowed to react for a further 12hours. The solution was then dialysed against excess water with numerouschanges, to remove free EDAC and the reaction by-products.

EXAMPLE 9 Steric Stabilization of Sigma Ludox as40 Silica Sol Using 95%poly[2-(dimethylamino)ethyl methacrylate]₁₀-Block-poly(ethylene oxide)₁₇Macro Raft Agent and 5% poly(2-(dimethylamino)ethylmethacrylate)₁₀-Block-poly(acrylamide)₂₅ Macro Raft Agent Part (a):Preparation of Sterically Stabilized Sigma Ludox AS30 Silica Particlesand a 95:5 Blend of the Macro-RAFT Agents of Example 8 Part (b) andExample 8 Part (d)

Ludox AS40 from Sigma Aldrich (5.0 g) was diluted with MQ water 100 g toyield a 2 wt % dispersion of the nanoparticles and the pH is 9.97. Amixture of macro-RAFT agents, which consisted of example 8 part (b)(1.72 g) and example 8 part (d) (0.13 g) was dissolved in 100 g of MQwater and the pH was 7.95. The 2 wt % dispersion was then poured intothe mixture of macro-RAFT agents. The mixture was vigorously stirred for5 hours at room temperature. The dispersion was then dialysed to removesalts, residual solvents, unwanted low molecular weight reaction sideproducts and unbound polymer. The solid content of the dialysed silicasol dispersion is 1.45%. The pH of sample was 7.65.

Part (b): Modification of Stabilisers of Silica Particles of Example 9Part (a)

To sterically stabilised silica sol particles prepared in example 9 part(a) (30 g), N-hydroxysuccinimide (NHS, 11.6 mg) and1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 16.6 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. 2,2′-(Ethylenedioxy)bis-(ethylamine) (45.1 mg) was thenadded to the reaction mixture and allowed to react for a further 12hours. The solution was then dialysed against excess water with numerouschanges, to remove free EDAC and the reaction by-products.

EXAMPLE 10 Steric Stabilization of 130 nm Silica Particles Usingpoly(2-(dimethylamino)ethyl methacrylate)₁₀-Block-poly(acrylamide)₂₅Macro Raft Agent Part (a): Silica Particles were Prepared Using theMethods of Costa et al

(Carlos A. R. Costa, Carlos A. P. Leite, and Fernando Galembeck J. Phys.Chem. B, 2003, 107 (20), 4747-4755.) to obtain 130 nm diameter silicaparticles at 0.18% solids in water.

Part (b): Steric Stabilization of the 130 nm Diameter Silica Particlesof Example 10 Part (a) Using the Macro-RAFT Agent of Example 8 Part (d)

Silica particle dispersion of example 10 part (a) (11.15 g) was dilutedwith MQ water (20 g) to yield a 0.1 wt % dispersion of the nanoparticleswith a pH of 9.26. Macro-RAFT agent of example 8 part (d) (0.023 g) wasdissolved in 25 g of MQ water (25 g) to yield a solution of pH 5.80. Thesilica dispersion and the macro-RAFT solution were then blended andvigorously stirred for 5 hours at room temperature. The dispersion wasthen centrifuged to remove salts, residual solvents, unwanted lowmolecular weight reaction side products and unbound polymer. The solidcontent of the sterically stabilised silica dispersion is 0.81%.

EXAMPLE 11 Steric Stabilization of the 130 nm Diameter Silica Particlesof Example 10 Part (a) Using the “Grow from” Approach

The silica particles of example 10 part (a) were RAFT functionalisedusing 6-(Triethoxysilyl)hexyl2-(((Methylthio)carbonothioyl)-2-phenylacetate and polymer chainscomprising poly methoxy-PEG acrylate (Aldrich 454 g/mol) were grown fromthe surface of the particles according to the methods of Ohno et al.(Kohji Ohno, Ying Ma, Yun Huang, Chizuru Mori, Yoshikazu Yahata,Yoshinobu Tsujii, Thomas Maschmeyer, John Moraes, and Sébastien PerrierMacromolecules, 2011, 44 (22), pp 8944-8953.) The molecular weightobtained for each anchored chain was approximately 56,000 g/mol. Thefinal particles were obtained in water at a solids content of 10 mg/mLand the particle size was 258 nm, as measured by DLS.

EXAMPLE 12 Steric Stabilization of 10-15 nm Gold Nanoparticles inAqueous Dispersion Using 95% poly(ethylene oxide)₁₇ Macro Raft Agent and5% poly(acrylamide)₂₀ Macro Raft Agent Part (a): Synthesis of 10-15 nmCitrate Stabilized Gold Nanoparticles Stable in Aqueous Medium

Citrate-stabilized gold nanoparticles (10-15 nm) were prepared usingFrens method (Frens, G. Nat. Phys. Sci. 1973, 241, 20-2.) Briefly, allglassware was first washed with an aqua regia solution (25 vol %concentrated nitric acid and 75 vol % concentrated hydrochloric acid),then rinsed with Milli-Q water several times, and dried. 100 ml of anaqueous solution containing tertrachloroaureic(III) acid trihydrate(0.01 g, 0.025 mmol) was refluxed in a 500 mL 3-necked round bottomflask. 2 ml solution of trisodium citrate dihydrate (0.02 g, 0.068 mmol)was added to it. The solution was heated to boiling point vigorousstirring. Boiling and vigorous stirring was maintained for 30 min. Aprogressive change of colour from yellow to wine red was observed. Thesolution was cooled down, dialysed to get rid of excess sodium citrateand stored in at 5° C. The nanoparticle concentration in the dispersionwas 50 ppm.

Part (b): Preparation of a poly(acrylamide)20 Macro-RAFT Agent Using:2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic Acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl} propanoic acid(0.71 g, 3.0 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.04 g, 0.15mmol), acrylamide (4.28 g, 60.2 mmol) in dioxane (7.5 g) and water (7.5g) was prepared in a 100 mL round bottom flask. This was stirredmagnetically and sparged with nitrogen for 15 minutes. The flask wasthen placed in a 70° C. oil bath with continued stirring for 4 hrs. Thepolymer solution had 25.17% solids.

Part (c): Preparation of Sterically Stabilized 10-15 nm GoldNanoparticles from the Citrate Stabilised Gold Nanoparticles of Example12 Part (a) and a 95:5 Blend of the Macro-RAFT Agent of Example 2 Part(a) and the Macro-RAFT Agent of Example 12 Part (b)

100 ml gold nanoparticle dispersion (50 ppm) of example 12 part (a) wastransferred to a 250 ml round bottom flask. A 10 ml solution containing0.012 g of the macro-RAFT agent of example 2 part (a) and 0.15 g themacro-RAFT agent of example 12 part (b) was then added. The mixture wasstirred vigorously with a magnetic stirrer bar for 2 hours at roomtemperature and then dialysed to remove salts, residual solvents,unwanted low molecular weight reaction side products and unboundpolymer. The purified nanoparticle dispersion was at a concentration of50 ppm and was stored in the fridge at 5° C. The resulting aqueousnanoparticle dispersion was found to be stable in phosphate buffersaline solution,

Part (d): Modification of Stabilisers for Gold Nanoparticles of Example12 Part (c)

Into coated nanoparticles prepared from example 12 part (c) (100 ml),N-hydroxysuccinimide (NHS, 4 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 4.1 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. A solution of diamine (21 mg of2,2′-(Ethylenedioxy)bis-(ethylamine) in 2 ml of water) was then added tothe reaction mixture and allowed to react for a further 3.5 hours. Thesolution was then dialysed against excess water with numerous changes,to remove free EDAC and the reaction by-products.

EXAMPLE 13 Steric stabilization of 3-8 nm gold nanoparticles dispersedin aqueous medium using thiol Modified poly(acrylamide)₂₀ Part (a):Thiol Modification of poly(acrylamide)20 Macro-RAFT Agent of PartExample 12 Part (b) Using isopropyl amine

A solution of the poly(acrylamide)20 macro-RAFT agent of example 12 part(b) (1 g, 0.6 mmol), isopropyl amine (1.77 g, 30 mmol) in dioxane (7.5g) and water (7.5 g) was prepared in a 100 mL round bottom flask. Thiswas stirred magnetically and sparged with nitrogen for 15 minutes, thenallowed to react for a 24 hours at 25° C. At the end of this period, thepolymer was precipitated in diethyl ether (50 ml). The precipitates wereseparated from the reaction mixture by filtration and dried under vacuumusing a rotary evaporator. The dried thiol terminated poly(acrylamide)20was sparged with nitrogen for 15 minutes and stored in an airtightcontainer at 20° C.

Part (b): Preparation of Sterically Stabilized 3-8 nm Gold Nanoparticlesin Aqueous Dispersion Using Thiol Modified poly(acrylamide)20 of Example13 Part (a)

Milli-Q water (250 mL) was refluxed in a 500 mL 3-necked round bottomflask. 25 mL of a aqueous solution containing tertrachloroaureic(III)acid trihydrate (0.0571 g, 0.1444 mmol) was then added and the solutionheated to boiling. Then, a solution in water (25 mL) of trisodiumcitrate dihydrate (0.5 g, 1.7 mmol) and thiol modifiedpoly(acrylamide)20 (0.12 g, 0.0779 mmol) of example 13 part (a) wasadded and the reaction carried out for 2 hours at 25° C. By the end ofthis period, the colour of the solution had turned from yellow to winered. The molar ratio of steric stabilizer to the tertrachloroaureic(III)acid trihydrate in this case is 0.5. The gold nanoparticles wereseparated from the dispersion by centrifugation at 52,000 g for 30 min.The nanoparticles were redispersed in Milli-Q water at a concentrationof 190 ppm. The size of gold nanoparticles obtained from TEM was 3-8 nm.

EXAMPLE 14 Steric Stabilization of 8-10 nm Gold Nanoparticles Dispersedin Aqueous Medium Using Thiol Modified poly(acrylamide)₂₀ of Example 13Part (a)

Milli-Q water (250 mL) was refluxed in a 500 mL 3-necked round bottomflask. 25 mL of an aqueous solution containing tertrachloroaureic(III)acid trihydrate (0.0652 g, 0.16 mmol) was added and the solution washeated to boiling. Then, a solution in water (25 mL) of trisodiumcitrate dihydrate (0.5 g, 1.7 mmol) and thiol modifiedpoly(acrylamide)20 (0.022 g, 0.0142 mmol) of example 13, part (a) wasadded and allowed to react for 2 hours at 25° C. By the end of thisperiod, the colour of the solution had turned from yellow to wine red.The molar ratio of steric stabilizer to the tertrachloroaureic(III) acidtrihydrate in this case was 0.09. The gold nanoparticles were separatedfrom the dispersion by centrifugation at 52,000 g for 30 min. Thenanoparticles were redispersed in Milli-Q water at a concentration of390ppm. The size of gold nanoparticles obtained from TEM was 8-10 nm.

EXAMPLE 15 Steric Stabilization of 30-40 nm Gold Nanoparticles Dispersedin Aqueous Medium Using Thiol Modified poly(acrylamide)₂₀ of Example 13Part (a) Part (a): Synthesis of 30-40 nm Citrate Stabilized GoldNanoparticles Stable in Aqueous Medium

Citrate-stabilized gold nanoparticles (30-40 nm) were prepared usingFrens method (Frens, G. Nat. Phys. Sci. 1973, 241, 20-2.) Briefly, allglassware was first washed with an aqua regia solution (25 vol %concentrated nitric acid and 75 vol % concentrated hydrochloric acid),then rinsed with Milli-Q water several times, and dried. 100 ml of anaqueous solution containing tertrachloroaureic(III) acid trihydrate(0.01 g, 0.025 mmo1) was refluxed in a 500 mL 3-necked round bottomflask. 1 ml solution of trisodium citrate dihydrate (0.01 g, 0.034 mmol)was then added. The solution was heated to boiling with vigorousstirring. Boiling and vigorous stirring was maintained for 30 min. Aprogressive change of colour from yellow to wine red was observed. Thesolution was cooled to ambient, dialysed to get rid of excess sodiumcitrate and stored at 5° C. The nanoparticle concentration in thedispersion was 50 ppm.

Part (b): Preparation of Sterically Stabilized 30-40 nm GoldNanoparticles from the Aqueous Gold Nanoparticle Dispersion of Example15 Part (a) and Thiol Modified poly(acrylamide)20 of Example 13 Part (a)

100 ml gold nanoparticle dispersion (50 ppm) of example 15 part (a) wastaken in a 250 ml round bottom flask. 10 ml solution of aqueous solutionof example 13 part (a) containing thiol modified poly(acrylamide)₂₀(0.0068 g, 0.0044 mmol) was then added. The mixture was stirredvigorously with a magnetic stirrer bar for 2 hours at room temperatureand then dialysed to remove salts, residual solvents, unwanted lowmolecular weight reaction side products and unbound polymer. Thepurified nanoparticle dispersion was then distilled to increase thesolids loading in the aqueous nanoparticle dispersion to 192ppm. Theresulting aqueous nanoparticle dispersion was found to be stable inphosphate buffer saline solution,

EXAMPLE 16 Synthesis of Polystyrene Nanoparticles in Aqueous DispersionUsing poly(styrene)₉-b-poly(acrylamide)₁₅ Macro Raft Agent Part (a):Preparation of Self Assembled poly(styrene)9-b-poly(acrylamide)15Macro-RAFT Agent Using:2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic Acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid(0.80 g, 3.36 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.10 g, 0.36mmol), acrylamide (3.71 g, 52.06 mmol) in dioxane (6.61 g) and water(4.41 g) was prepared in a 50 mL round bottom flask. This was stirredmagnetically and sparged with nitrogen for 10 minutes. The flask wasthen heated at 70° C. for 5 hrs to produce the clear homopolymersolution. At the end of this period, styrene (3.16 g, 30.3 mmol),4,4′-azobis(4-cyanovaleric acid) (0.19 g, 0.69 mmol), dioxane (21.15 g)and water (6.14 g) were added to the flask. The mixture was stirred,deoxygenated with nitrogen for 10 minutes. The flask was then immersedback in an oil bath at 70° C. for overnight with constant stirring.

Part (b): Synthesis of Polystyrene Nanoparticles Using the SelfAssembled Macro-RAFT Agent Prepared in Example 16 Part (a)

To a clear dispersion of macro-RAFT agent from example 16 part (a) (1.00g) in a 50 mL round bottom flask on a magnetic stirrer, sodium hydroxidesolution (1.94 g of 0.3% solution, 0.15 mmol with water (22.1 g)) wasadded drop wise. To this mixture styrene (1.109 g, 10.5 mmol) was addedand stirred overnight. 4,4′-azobis(4-cyanovaleric acid) (15.5 mg, 0.055mmol) and sodium hydroxide solution (1.04 g of 3% solution, 0.78 mmol)were added. The flask was stirred for 2 hours, then sealed andsubsequently deoxygenated with nitrogen sparging for 10 minutes. Thewhole flask was immersed in an oil bath with a temperature setting of80° C. and maintained at that temperature for 5 hours under constantmagnetic stirring. The latex contained particles with average diameterof 15 nm by Zetasizer light scattering. The latex was dialysed againstmilli-Q water to remove impurities.

EXAMPLE 17 Synthesis of Polystyrene Nanoparticles in Aqueous DispersionUsing Self Assembled poly(styrene)₉-b-poly(acrylamide)₂₀ Macro RaftAgent of Example 16 Part (a) Part (a): Further Growth of the SelfAssembled Macro-RAFT Agent of Example 16 Part (a) to Formpoly(styrene)52-b-poly(acrylamide)20 Macro-RAFT

To a clear dispersion of macro-RAFT agent from example 16 part (a) (1.02g) in a 25 mL round bottom flask on a magnetic stirrer, sodium hydroxidesolution (0.44 g of 3% solution, 0.33 mmol), 4,4′-azobis(4-cyanovalericacid) (14.1 mg, 0.05 mmol) and water (14.0 g) were added and stirred todissolved. To this mixture styrene (0.61 g, 5.85 mmol) was added andstirred overnight. The flask was then sealed and subsequentlydeoxygenated with nitrogen sparging for 10 minutes. The whole flask wasimmersed in an oil bath with a temperature setting of 70° C. andmaintained at that temperature for 6 hours under constant magneticstirring. A clear dispersion was obtained

Part (b): Synthesis of Polystyrene Nanoparticles Using the Macro-RAFTAgent Dispersion Prepared in Example 17 Part (a)

To a clear solution of macro-RAFT agent from example 17 part (a) (6.09g) in a 50 mL round bottom flask on a magnetic stirrer, sodium hydroxidesolution (0.36 g of 3% solution, 0.27 mmol), 4,4′-azobis(4-cyanovalericacid) (26.6 mg, 0.095 mmol), styrene (0.45 g, 4.37 mmol) and water (8.31g) were added. The flask was stirred for 5 hours, then sealed andsubsequently deoxygenated with nitrogen sparging for 10 minutes. Thewhole flask was immersed in an oil bath with a temperature setting of70° C. and maintained at that temperature for overnight under constantmagnetic stirring. The latex contained particles with mean diameter of47 nm by Zetasizer light scattering. The latex was dialysed againstmilli-Q water to remove impurities.

EXAMPLE 18 Synthesis of Polystyrene Nanoparticles in Aqueous DispersionUsing poly(acrylamide)₂₀ Macro Raft Agent Part (a): Preparation ofpoly(acrylamide)20 Macro-RAFT Agent Using:2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic Acid

A solution of 2-{[(butylsulfanyl)carbonothioyl]sulfanyl}propanoic acid(0.73 g, 3.08 mmol), 4,4′-azobis(4-cyanovaleric acid) (0.07 g, 0.3mmol), acrylamide (4.30 g, 60.5 mmol) in dioxane (15 g) and water (7.5g) was prepared in a 100 mL round bottom flask. This was stirredmagnetically and sparged with nitrogen for 15 minutes. The flask wasthen heated at 70° C. for 4 hrs to produce the clear homopolymersolution.

Part (b): Synthesis of Polystyrene Nanoparticles Using the Macro-RAFTAgent Prepared in Example 18 Part (a)

A clear solution of macro-RAFT agent from example 18 part (a) (1.05 g),sodium hydroxide (2.07 g of 3% solution, 1.55 mmol) and water (12.16 g)was prepared in a 25 mL round bottom flask, stirring on a magneticstirrer. To this solution 4,4′-azobis(4-cyanovaleric acid) (13.6 mg,0.049 mmol), dioxane (1.1 g) and styrene (1.125 g, 10.8 mmol) wereadded. The mixture was stirred for 2 hours to obtain an emulsion likemixture. The flask was sealed and subsequently deoxygenated withnitrogen sparging for 10 minutes. The whole flask was immersed in an oilbath with a temperature setting of 70° C. and maintained at thattemperature for overnight under constant magnetic stirring. The latexcontained particles with average diameter of 200 nm by Zetasizer lightscattering. The latex was dialysed against milli-Q water to removeimpurities.

EXAMPLE 19 Stabilisation of Iron Oxide Nanoparticles with Dextran fromleuconostoc mesenteroides (Average Molecular Weight of 9000-11,000,Sigma Aldrich) Coated Particles. (Example 19 is a Comparative Example)

25 ml of 0.5 M FeCl₂/4H₂O and 25 ml of 1M FeCl₃/6H₂O were mixed andmagnetically stirred in a 500 ml 3 neck round bottom flask. Theresulting solution was diluted by adding 100 ml of MQ water and placedin an oil bath at 70° C. Dextran solution (50 ml of 15% solids in water)was added and the solution maintained in the oil bath for 10 minutes.Ammonia solution (30 ml, 28%) was then added and the mixture kept at 70°C. for a further 45 minutes. The reaction product was cooled to roomtemperature and dialysed against MQ water to remove excess ammonia. Thewater was changed at least three times. Larger aggregates were removedby magnetic sedimentation. Volume was reduced to about 100 ml byremoving water on rotary evaporator. The final dispersion was sonicatedat 70% AMP using an ultrasonicator for 10 minutes and at also at 30% AMPfor 30 minute.

EXAMPLE 20 Steric Stabilization of Iron Oxide Nanoparticles of Example 1Part (a) Using 50% poly(monoacryloxyethylphosphate)₁₀-Block-poly(ethylene oxide)₁₇ Macro Raft Agent of Example 2Part (b) and 50% Amine Modified poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ Macro Raft Agent Part (a):Preparation of Sterically Stabilized Iron Oxide Nanoparticles from theAqueous Ferrofluid of Example 1 Part (a) and a 50:50 Blend of theMacro-RAFT Agent of Example 2 Part (b) and the Macro-RAFT Agent ofExample 2 Part (c)

Aqueous ferrofluid prepared according to example 1, part (a) (8.10 g)was diluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this prepared nanoparticle dispersion was thenraised to 5. A blend of macro-RAFT which consist of 50 g of at 5.1 wt %solids, 3.3 wt % of which was the macro-RAFT agent of example 2 part (b)and 1.8 wt % of which was the macro-RAFT agent of example 2 part (c)were mixed together and the pH adjusted to 5 using 0.1M NaOH. Thedispersion of iron oxide, maintained at the same pH was then added tothe macro-RAFT blend. The mixture was vigorously stirred for 2 hours atroom temperature before the pH was adjusted to 7.0. The mixture was thenleft stirring for another 12 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solid content ofthe dialysed aqueous ferrofluid dispersion is 0.6%. Part (f):Modification of stabilisers for iron oxide particles of example 20 part(a)

Into coated nanoparticles prepared from example 20 part (a) (70 g),N-hydroxysuccinimide (NHS, 89.3 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 127 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. 291 mg of 2,2′-(Ethylenedioxy)bis-(ethylamine) was thenadded to the reaction mixture and allowed to react for a further 12hours. The solution was then dialysed against excess water with numerouschanges, to remove free EDAC and the reaction by-products.

EXAMPLE 21 Steric Stabilization of Iron Oxide Nanoparticles of Example 1Part (a) Using 80% poly(monoacryloxyethylphosphate)₁₀-Block-poly(ethylene oxide)₁₇ Macro Raft Agent of Example 2Part (b) and 20% Amine Modified poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ Macro Raft Agent Part (a):Preparation of Sterically Stabilized Iron Oxide Nanoparticles from theAqueous Ferrofluid of Example 1 Part (a) and a 80:20 Blend of theMacro-RAFT Agent of Example 2 Part (b) and the Macro-RAFT Agent ofExample 2 Part (c)

Aqueous ferrofluid prepared according to example 1, part (a) (8.10 g)was diluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this prepared nanoparticle dispersion was thenraised to 5. A blend of macro-RAFT which consist of 50 g of at 6.0 wt %solids, 5.28 wt % of which was the macro-RAFT agent of example 2 part(b) and 0.72 wt % of which was the macro-RAFT agent of example 2 part(c) were mixed together and the pH adjusted to 5 using 0.1M NaOH. Thedispersion of iron oxide, maintained at the same pH was then added tothe macro-RAFT blend. The mixture was vigorously stirred for 2 hours atroom temperature before the pH was adjusted to 7.0. The mixture was thenleft stirring for another 12 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solid content ofthe dialysed aqueous ferrofluid dispersion is 0.7%.

Part (b): Modification of Stabilisers for Iron Oxide Particles ofExample 21 Part (a)

Into coated nanoparticles prepared from example 2, Part (e) (60 g),N-hydroxysuccinimide (NHS, 39.4 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 56.2 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. 130 mg of 2,2′-(Ethylenedioxy)bis-(ethylamine) was thenadded to the reaction mixture and allowed to react for a further 12hours. The solution was then dialysed against excess water with numerouschanges, to remove free EDAC and the reaction by-products.

EXAMPLE 22 Steric Stabilization of Iron Oxide Nanoparticles of Example 1Part (a) Using 90% poly(monoacryloxyethylphosphate)₁₀-Block-poly(ethylene oxide)₁₇ Macro Raft Agent of Example 2Part (b) and 10% Amine Modified poly(monoacryloxyethylphosphate)₁₀-Block-poly(acrylamide)₂₀ Macro Raft Agent Part (a):Preparation of Sterically Stabilized Iron Oxide Nanoparticles from theAqueous Ferrofluid of Example 1 Part (a) and a 90:10 Blend of theMacro-RAFT Agent of Example 2 Part (b) and the Macro-RAFT Agent ofExample 2 Part (c)

Aqueous ferrofluid prepared according to example 1, part (a) (8.10 g)was diluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this prepared nanoparticle dispersion was thenraised to 5. A blend of macro-RAFT which consist of 50 g of at 6.3 wt %solids, 6.4 wt % of which was the macro-RAFT agent of example 2 part (b)and 05.9 wt % of which was the macro-RAFT agent of example 2 part (c)were mixed together and the pH adjusted to 5 using 0.1M NaOH. Thedispersion of iron oxide, maintained at the same pH was then added tothe macro-RAFT blend. The mixture was vigorously stirred for 2 hours atroom temperature before the pH was adjusted to 7.0. The mixture was thenleft stirring for another 12 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solid content ofthe dialysed aqueous ferrofluid dispersion is 0.87%.

Part (b): Amine Modification of Stabilisers for Iron Oxide Particles ofExample 22 Part (a

Into coated nanoparticles prepared from example 2 part (a) (60 g),N-hydroxysuccinimide (NHS, 24.7 mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 34 mg) were added,mixed by shaking and allowed to react for 2 hours at room temperature.18.2 mg of 2,2′-(Ethylenedioxy)bis-(ethylamine) was then added to thereaction mixture and allowed to react for a further 12 hours. Thesolution was then dialysed against excess water with numerous changes,to remove free EDAC and the reaction by-products.

EXAMPLE 23 Stabilisation of Iron Oxide Nanoparticles with leuconostocmesenteroides dextran (Average Molecular Weight 35,000-45,000 from SigmaAldrich) (Example 23 is a Comparative Example)

25 ml of 0.5 M FeCl₂/4H₂O in solutions and 25 ml of 1M FeCl₃/6H₂O insolution was magnetically stirred in a 500 ml 3 neck round bottom flask.The solution mixture was diluted by adding 100 ml of Mili-Q water andthe resulting solution placed in an oil bath at 70° C. After 10 minutesdextran solution (15%, 50 ml) was then added followed by ammoniasolution (28% 30 ml). The mixture was kept at 70° C. for a further 45minutes. The reaction mixture was cooled to room temperature anddialysed against MQ water to remove excess ammonia. The water waschanged at least three times. Larger aggregates were removed by magneticsedimentation. The volume of the dispersion was reduced to about 100 mlby using a rotary evaporator. The final dispersion was sonicated at 70%AMP using an ultrasonicator for 10 minutes followed by sonication at 30%AMP for 30 minute.

EXAMPLE 24 Steric Stabilization of Iron Oxide Nanoparticles of Example 1Part (a) Using 98% poly(monoacryloxyethylphosphate)₁₀-Block-poly(ethylene oxide)₁₇ Macro Raft Agent and 2% AmineModified poly(monoacryloxyethyl phosphate)₁₀-Block-poly(acrylamide)₂₀Macro Raft Agent Part (a): Preparation of Sterically Stabilized IronOxide Nanoparticles from the Aqueous Ferrofluid of Example 1 Part (a)and a 98:2 Blend of the Macro-RAFT Agent of Example 2 Part (b) and theMacro-RAFT Agent of example 2 Part (c). [EP341063]

Aqueous ferrofluid prepared according to example 1, part (a) (8.10 g)was diluted with MQ water (50 g) to yield a 0.5 wt % dispersion of thenanoparticles. The pH of this prepared nanoparticle dispersion was thenraised to 5. A blend of macro-RAFT which consist of 50 g of at 6.48 wt %solids, 6.4 wt % of which was the macro-RAFT agent of example 2 part (b)and 0.08 wt % of which was the macro-RAFT agent of example 2 part (c)were mixed together and the pH adjusted to 5 using 0.1M NaOH. Thedispersion of iron oxide, maintained at the same pH was then added tothe macro-RAFT blend. The mixture was vigorously stirred for 2 hours atroom temperature before the pH was adjusted to 7.0. The mixture was thenleft stirring for another 12 hours. At this pH the copolymer remainedpartially neutralized while the nanoparticles were sufficiently abovetheir point of zero charge to also be stable. The dispersion was thendialysed to remove salts, residual solvents, unwanted low molecularweight reaction side products and unbound polymer. The solid content ofthe dialysed aqueous ferrofluid dispersion is 0.8%.

Part (b): Amine Modification of Stabilisers of Iron Oxide Particles ofExample 24 Part (a)

Into coated nanoparticles prepared from example 24, Part (a) (55 g),N-hydroxysuccinimide (NHS, 5.1′mg) and then1-Ethyl-3-(3-Dimethylamino-propyl)carbodiimide (EDAC, 6.8 mg) wereadded, mixed by shaking and allowed to react for 2 hours at roomtemperature. 2,2′-(Ethylenedioxy)bis-(ethylamine) (18.2 mg) was thenadded to the reaction mixture, which was allowed to react for a further12 hours. The solution was then dialysed against excess water withnumerous changes, to remove free EDAC and the reaction by-products.

EXAMPLE 25 General Method for Preparation of Spheroids

Human DLD-1 colon cancer cells and human PA-1 ovarian cancer cells wereobtained from the American Type Culture Collection (Manassas, Va., USA).Cells were maintained in complete media (Advanced DMEM (Invitrogen) andsupplemented with 2% foetal bovine serum (Sigma) and 2mM Glutamax™(Invitrogen)) at 37° C. in a humidified, 5% CO₂ atmosphere. Spheroidswere formed by plating 1.5×10⁵ cells/ml onto agarose coated 96 wellimaging plates (BD Biosciences) and the cells allowed to aggregate for72 hrs at 37° C. in a humidified, 5% CO₂ atmosphere resulting in theformation of single spheroid per well.

EXAMPLE 26 Assessment of Cytotoxicity of Active Compounds andNanoparticles

Active compounds and/or nanoparticles were diluted as required in cellmedia immediately prior to the assay. Cytotoxicity was determined usingthe MTT assay as follows. 1×10⁵ cells were seeded onto each well of flatbottomed 96-well plates and allowed to attach overnight. Solutions ofcompounds +/−nanoparticles were added to triplicate wells atconcentrations spanning a 4-log range and incubated for 72 hrs. MTT(3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (1.0 mM)was added to each well and were incubated for a further 4 hrs. Theculture medium was removed from each well, DMSO (150 μL) was added, theplate shaken for 5 seconds and the absorbance measured immediately at600 nm in a Victor³V microplate reader (Perkin Elmer). IC₅₀ values weredetermined as the drug concentration that reduced the absorbance to 50%of that in untreated control wells. At least three independentexperiments were performed for each compound with triplicate readings ineach experiment. Cytotoxicity values for all active compounds used arelisted in Table 2.

EXAMPLE 27 General Method for Treating the Cancer Spheroids of Example 5with (a) Nanoparticles Alone, (b) Co-Administration of Nanoparticles andActive Compound or (c) Time Course Treatment of Nanoparticle First thenActive Compound

All nanoparticles were sterilised either by filtration through a 0.22 μmfilter or by autoclaving at 120° C., 2 KPa for 20 min in a Tomy highpressure steam sterilizer ES-315 before use in cellular assays.

-   -   (a) To the suspension of the 3 day old spheroids from example        25, 100 μl of a solution containing nanoparticles incomplete        media was added to each spheroid, to yield a final concentration        of particles of 10 ppm in 200 μl total volume. he spheroids were        replaced in an incubator at 37° C., 5% CO₂. After 24 hours        incubation, the nanoparticles in the media were removed by        washing with excess phosphate buffered saline prior to further        experimentation.    -   (b) To the suspension of the 3 day old spheroids from example        25, 100 μl of a solution containing active compound and        nanoparticles incomplete media was added to each spheroid, to        yield a final concentration of particles of 10 ppm. The        concentration of the active compounds used is defined in        Table 3. The spheroids were replaced in an incubator 37° C., 5%        CO₂ atmosphere. After 24 hours incubation, the free active        compound and nanoparticles in the media were removed by washing        with excess phosphate buffered saline.    -   (c) To the suspension of the 3 day old spheroids from example        25, 100 μl of a solution containing nanoparticles in complete        media was added to each spheroid, to yield a final concentration        of particles of 10 ppm. The spheroids were replaced in an        incubator at 37° C., 5% CO₂. After 24 hours incubation, the        spheroids were dosed with an active compound at the        concentration listed in Table 2 and incubated for a further 24        hours at 37° C., 5% CO₂. The free active compound and        nanoparticles in the media were removed by washing with excess        phosphate buffer saline prior to further experimentation.

EXAMPLE 28 General Method for Imaging Spheroids Treated withNanoparticles and a Fluorescent Active Compound by Confocal Microscopy

Spheroids from example 25 were treated as per example 27b and 27c thentransferred to a glass bottomed 35 mm dish (Mattek) and imaged on anOlympus FV1000 confocal microscope using an Olympus UPLAPO 10×/0.40 airobjective lens. Single confocal images through the central region of thespheroid were taken. Excitation and emission settings were fluorophoredependent: Doxorubicin ex:559 nm em:575-675; Mitoxantrone ex:405 nm,em:575-675

EXAMPLE 29 General Method for Measuring Effectiveness of Active Compound+/− Nanoparticles in Spheroids (Outgrowth Assay)

Spheroids from example 25 were treated as per example 27b and 27c. Thespheroids were then transferred to a 24 well plate using a wide boretransfer pipette and the medium replaced with 1 mL of fresh media ineach well. The spheroids were then incubated for 48 hours at 37° C. in a5% CO₂ humidified environment, allowing the spheroid to attach to theplate and the cells to grow out from the spheroid onto the surface ofthe plate. Hoechst 33342 was then added to the wells and incubated for30 minutes at 37° C. in a 5% CO₂ humidified environment. Widefieldfluorescence images of the brightfield and Hoechst 33342 stained nucleiwere taken of the cells that had grown out from the spheroid (OlympusCellR). To quantitate the outgrowth, the number of nuclei within a 60°angle from the edge of the spheroid was counted. These values were thenplotted in a graph normalised to spheroids treated with active compoundalone or untreated control spheroids for comparison.

EXAMPLE 30 Sterically Stabilised Nanoparticles are Able to Penetrateinto Spheroids

Spheroids from example 25 were treated as per example 27a with particlesfrom example 2 and washed with phosphate buffered saline, followed byprimary fixation with 2.5% glutaraldehyde solution and secondaryfixation with 1% osmium tetroxide. The spheroids were washed thendehydrated in a gradient of ethanol and infiltrated with Spurr's Resin.Ultra-thin sections with a nominal thickness of 95 nm were cut, placedon mesh grids and post stained with uranyl acetate and lead citrate. TEMimages of the spheroid sections were obtained using a JEOL 1400 TEM at120 kV.

The images in FIG. 1 were taken from the central region of the spheroidand show an accumulation of nanoparticles (darker stained areas asindicated with arrows) within the cytoplasm of the cells. The enlargedregion indicated by the box shows the well dispersed individualnanoparticles.

EXAMPLE 31 The effect of Nanoparticles on Drug Diffusion

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27a with nanoparticles from examples 2, 3, and 5)and imaged under conditions described in example 28. Doxorubicin alonediffused approximately 70 μm into the spheroid. Co-administration ofdoxorubicin and NP3 or 5 enhanced the spheroid penetration ofdoxorubicin to approximately 100 μm. In contrast, co-administration ofNP2 and doxorubicin resulted in doxorubicin diffusion throughout theentire spheroid (FIG. 2A). Mitoxantrone alone also diffusedapproximately 70 μm into the spheroid. Co-administration of NP3 andmitoxantrone had little effect on mitoxantrone diffusion, whereasco-administration of mitoxantrone and NP2 or NP5 significantly enhancedthe diffusion of mitoxantrone into the spheroid (FIG. 2B).

EXAMPLE 32 The Effect of Nanoparticles on Spheroid Viability

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27a with nanoparticles from examples 1, 2, 4, 8, 9,12, 13, 15, 16, and 18. The effectiveness of nanoparticles alone inspheroids was assessed as per example 29. It was found that the majorityof nanoparticles tested had little cytotoxic effect as shown in FIG. 3.

EXAMPLE 33 The effect of Nanoparticles with Different Core Types on theViability of Spheroids when Co-Administered With Doxorubicin

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27b with nanoparticles from examples 2, 4, 6, 7, 9,10, 11, 12, 13, 14, 16, 17, and 18 and doxorubicin. Effectiveness wasdetermined as per example 29. FIG. 4 shows that co-administration ofnanoparticles NP2 (iron core), NP11(silica core), NP12 (gold core), andNP18 (polystyrene core) with doxorubicin was more effective thandoxorubicin treatment alone as shown by the decreased cellular outgrowthfrom the spheroids. The composition of the nanoparticle core does notcorrelate with effectiveness.

EXAMPLE 34 The Effect of Nanoparticles with Different Core Sizes on theViability of Spheroids when Co-Administered with Doxorubicin

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27b with nanoparticles from examples 1, 2, 4, 7, 9,10, 11, 12, 13, 14, 16, 17, and 18 and doxorubicin. Effectiveness wasdetermined as per example 29. Several different nanoparticles with arange of core sizes from 10 nm to 200 nm when co-administered withdoxorubicin were shown to be more effective than doxorubicin alone (FIG.5). It was shown that co-administration of particles NP1, NP2, NP12 andNP18 co-administered with doxorubicin was approximately 50% moreeffective than doxorubicin treatment alone.

EXAMPLE 35 The effect of the Functionalised Stabiliser End Group onSpheroid Viability when Co-Administered with Doxorubicin

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27b with nanoparticles listed in examples 2, 3, 4,5, 20, 21, 22, and 24 and doxorubicin. Effectiveness was determined asper example 29. It was found that the amine functionalised end groupeffected spheroid viability when co-administered with doxorubicin. Byvarying the percentage of amine functionalised groups on the surface ofthe nanoparticles, we found that particles containing between 5-20%amine functionalised end groups were the most effective whenco-administered with doxorubicin. Doxorubicin was the most effectivewhen co-administered with nanoparticles containing stabilisers with 5%amine functionalised end groups.

EXAMPLE 36 Nanoparticles of Different Cores Stabilised with 5% AmineFunctionalised End Groups Co-Administered to Spheroids with Doxorubicin

DLD-1 spheroids prepared as per example 25 were dosed as per theprotocol in example 27b with nanoparticles listed in examples 2, 8, 9and 12 and doxorubicin. Effectiveness was determined as per example 29.Nanoparticles stabilised with 5% amine functionalised end groups weremade with different cores and it was shown that all were more effectivethan doxorubicin alone and had a similar level of effectiveness whenco-administered with doxorubicin (FIG. 7).

EXAMPLE 37 The Effect of the Active Compounds when Co-Administered withNanoparticles on the Viability of Spheroids Made from Two DifferentCancer Cell Lines

DLD-1 and PA-1 spheroids were prepared as per example 25 and dosed asper the protocol in example 27b with nanoparticles listed in examples 2,3, 4, and 5 and active compounds. Effectiveness was determined as perexample 29. The majority of particles and active compounds had similareffectiveness between the two cell lines, with the exception ofmitoxantrone. Co-administration of mitoxantrone and nanoparticles wassignificantly more effective in the PA-1 cell ovarian cancer cell linecompared to the DLD-1 colorectal cancer line.

EXAMPLE 38 Comparative Example Between Co-Administration ofNanoparticles and Active Compounds, and Administration of Nanoparticleswith Delayed Administration of Active Compounds in Two Different CellLines

DLD-1 and PA-1 spheroids were prepared as per example 25 and dosed asper the protocol in example 27b and 27c with the nanoparticles listed inexamples 2, 3, 4, and 5 and active compounds. Effectiveness wasdetermined as per example 29. FIGS. 9 and 10 show that for some particleand active combinations e.g. 5FU+NP3 there is no difference ineffectiveness in either cell line for either mode of treatment. Ingeneral however, there is little correlation between treatment scheduleand effectiveness between the two cell lines tested. It will beimportant to determine which nanoparticle/active combination is mosteffective for each cancer type. It should be noted that mitoxantronerequires the co-administration of nanoparticles in PA-1 cells forgreatest effectiveness.

EXAMPLE 39 Examples of the Most Effective Co-Administered Combination ofNanoparticles and Active Compound for Each Active Compound Tested

DLD-1 and PA-1 spheroids were prepared as per example 25 and dosed asper the protocol in example 27b with the nanoparticles listed inexamples 2, 5, 14, 20, 21, and 22 and active compounds. Effectivenesswas determined as per example 29. The results presented are for the mosteffective nanoparticle(s) co-administered with each active compound inboth DLD-1 spheroids (FIG. 11A) and PA-1 spheroids (FIG. 11B).

EXAMPLE 40 Treating the Cancer Spheroids of Example 25 with the IronOxide Nanoparticles of Examples 1 and 2 to Enable Spheroid Penetrationby Cisplatin

DLD-1 spheroids prepared as per example 25 were dosed as with 100 μl ofsolution of complete media containing cisplatin and iron oxidenanoparticles from examples 1 and 2 to yield a final concentration ofboth cisplatin and iron oxide of 6 ppm. The spheroids with iron oxideparticles and cisplatin were replaced in the incubator and maintained at37° C. in a 5% CO₂ atmosphere. After 48 hours incubation, the freecisplatin and nanoparticles in the media were washed with excessphosphate buffered saline. Analysis by atomic absorption spectroscopyshowed that after 48 hours incubation the concentration of cisplatin inthe spheroids with NP1 nanoparticles, NP2 nanoparticles and without ironoxide particles was 0.60, 0.63 and 0.20 ppb, respectively, a 3-foldincrease in cisplatin accumulation when nanoparticles were present.

EXAMPLE 41 Comparative Example: Doxorubicin Penetration into Spheroidswhen Co-Administered with Anchored Sterically Stabilised ParticlesCompared to Co-Administration with Unanchored Sterically StabilisedParticles

DLD-1 spheroids from example 25 were either dosed with NP2 (example 2),which are particles coated with a stabiliser containing a phosphateanchoring group or NP19 or NP23 (examples 19 and 23), which areparticles coated with a stabiliser that has no anchoring portion as perexample 27b. The spheroid was then imaged by confocal microscopy (as perexample 28) to visualise doxorubicin fluorescence. Spheroids treatedwith doxorubicin and NP2 had significantly more doxorubicin fluorescencein the centre of the spheroid compared to the spheroids treated withdoxorubicin alone and to spheroids treated with doxorubicinco-administered with the unanchored sterically stabilised particles NP19and NP23 (FIG. 12).

EXAMPLE 42 Potential Testing Regime to Determine the Most EffectiveNanoparticle and Active Compound for Patient Tumours

To identify which type(s) of nanoparticles and which type(s) of activedrug and an optimum combination of nanoparticles and drug were the mosteffective for an individual patient, tumour biopsies would initially betested. Several core tumour biopsies would be taken from a patient,dissected into smaller samples (approx 1 mm³) and dosed with selectednanoparticle/drug combinations. Each dosed sample would be flanked by anuntreated sample and a drug only to control for intra-tumourvariability. After 24 hrs, the sample would be subjected to an outgrowthassay to measure the efficacy of the tumour treatments withnanoparticles/drug to determine the most effective composition andadministration of nanoparticles and drug.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 1 LIST OF NANOPARTICLES USED TO EXEMPLIFY THIS PATENT Example NPsNP core, diameter Coating type 1 NP1 Fe₂O₃, 10-15 nm 100% diCOO 2 NP2Fe₂O₃, 10-15 nm 95% PEG 5% NH₂ 3 NP3 Fe₂O₃, 10-15 nm 100% PEG(CH₃) 4 NP4Fe₂O₃, 10-15 nm 100% COO 5 NP5 Fe₂O₃, 10-15 nm 100% NH₂ 6 NP6 Fe₂O₃,10-15 nm 95% COO 5% NH₂ 7 NP7 Fe₂O₃, 30-40 nm 100% COO 8 NP8 SiO₂, 10-15nm 95% PEG 5% NH₂ 9 NP9 SiO₂, 30-40 nm 95% PEG 5% NH₂ 10 NP10 SiO₂, 130nm 100% COO 11 NP11 SiO₂, 130 nm 100% PEGAcrylate 12 NP12 Gold, 10-15 nm95% PEG 5% NH₂ 13 NP13 Gold, 3-8 nm 100% COO 14 NP14 Gold, 10-15 nm 100%COO 15 NP15 Gold, 30-40 nm 100% COO 16 NP16 PSty, ~15 nm 100% COO 17NP17 PSty, ~40 nm 100% COO 18 NP18 PSty, ~200 nm 100% COO 19 NP19 Fe₂O₃,10-15 nm 10K Dextran 10 NP20 Fe₂O₃, 10-15 nm 50% PEG 50% NH₂ 21 NP21Fe₂O₃, 10-15 nm 80% PEG 20% NH₂ 22 NP22 Fe₂O₃, 10-15 nm 90% PEG 10% NH₂23 NP23 Fe₂O₃, 10-15 nm 40K Dextran 24 NP24 Fe₂O₃, 10-15 nm 98% PEG 2%NH₂

TABLE 2 72 hr IC₅₀ values for the active compounds used in this study.Active Compound DLD-1 PA-1 Doxorubicin (Dox) 1 μM 1 μM Cisplatin (Cis)10 μM 0.6 uM Mitoxantrone (Mito) 40 nM 20 nM Paclitaxel (Pac)indeterminable* indeterminable* 5-Fluorouracil (5FU) 10 μM 10 μM*Paclitaxel was cytotoxic at concentrations as low as 0.1 nM in thisassay.

TABLE 3 Concentrations of active compounds used for dosing in each cellline. Active Compound DLD-1 PA-1 Doxorubicin (Dox) 1 μM 1 μM Cisplatin(Cis) 10 μM 2 μM Mitoxantrone (Mito) 30 nM 30 nM Paclitaxel (Pac) 10 nM10 nM 5-Fluorouracil (5FU) 10 μM 10 μM

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1. A method of treating a solid tumour in a subject, said methodcomprising co-administering to said subject an effective amount ofparticulate material and a cellular toxin for a time and underconditions sufficient to facilitate distribution of said particulatematerial and toxin to said tumour wherein: (i) said particulate materialis administered in the form of a dispersion in a liquid carrier, theparticulate material being maintained in the dispersed state by astabiliser; and (ii) said stabiliser comprising an anchoring portionthat (a) anchors the stabiliser to the particulate material, and (b) isdifferent from the remainder of the stabiliser; and wherein saidparticulate material and toxin penetrate said solid tumour.
 2. Use ofparticulate material and a cellular toxin in the manufacture of amedicament for the treatment of a solid tumour wherein: (i) saidparticulate material is in the form of a dispersion in a liquid carrier,the particulate material being maintained in the dispersed state by astabiliser; and (ii) said stabiliser comprising an anchoring portionthat (a) anchors the stabiliser to the particulate material, and (b) isdifferent from the remainder of the stabiliser; and wherein saidparticulate material and toxin penetrate said solid tumour.
 3. Themethod according to claim 1 or the use according to claim 2, wherein thestabiliser is a steric stabiliser comprising a steric stabilisingpolymeric segment and an anchoring portion, wherein the stericstabilising polymeric segment is different from the anchoring portion,and wherein the anchoring portion anchors the stabiliser to theparticulate material.
 4. The method or use according to claim 3, whereinthe steric stabilising polymeric segment comprises a terminal ionicfunctional group.
 5. The method or use according to claim 4, wherein theionic functional group is a cation.
 6. The method or use according toany one of claims 3 to 5, wherein the steric stabilising polymericsegment of the stabiliser comprises polymer selected frompoly(acrylamide), poly(ethylene oxide), poly(hydroxyethylacrylate),poly(N-isopropylacrylamide), poly(dimethylaminoethyl methacrylate),poly(vinyl pyrrolidone), and copolymers thereof.
 7. The method or useaccording to any one of claims 1 to 6, wherein the anchoring portioncomprising one or more carboxylic acid groups, one or more phosphategroups, one or more phosphinate groups, one or more thiol groups, one ormore thiocarbonylthio groups, one or more sulfonic acid groups,ethoxysilyl groups, or combinations thereof.
 8. The method or useaccording to any one of claims 1 to 7, wherein the stabiliser is asteric stabiliser comprising a steric stabilising polymeric segment andan anchoring polymeric segment.
 9. The method or use according to claim8, wherein one or both of the steric stabilising and anchoring polymericsegments comprise the polymerised residue of one or more ethylenicallyunsaturated monomers.
 10. The method or use according to any one ofclaims 1 to 9, wherein the stabiliser has a number average molecularweight of less than about 30,000.
 11. The method or use according to anyone of claims 1 to 10, wherein the particulate material is selected froma metal, a metal alloy, a metal salt, a metal complex, a metal oxide, aninorganic oxide, a radioactive isotope, a polymer particle, andcombinations thereof.
 12. The method or use according to any one ofclaims 1 to 11, wherein the particulate material ranges in size fromabout 10 nm to about 350 nm.
 13. The method or use according any one ofclaims 1 to 12, wherein said particulate material is iron oxide rangingin size from about 10 nm to about 15 nm.
 14. The method or use accordingany one of claims 1 to 12, wherein said particulate material is goldranging in size from about 10 nm to about 15 nm.
 15. The method or useaccording any one of claims 1 to 12, wherein said particulate materialis silicon oxide ranging in size from about 10 nm to about 15 nm. 16.The method or use according any one of claims 1 to 12, wherein saidparticulate material is silicon oxide ranging in size from about 30 nmto about 40 nm.
 17. The method or use according any one of claims 1 to12, wherein said particulate material is polystyrene ranging in sizefrom about 10 nm to about 15 nm.
 18. The method or use according to anyone of claims 1 to 17, wherein said solid tumour is benign.
 19. Themethod or use according to any one of claims 1 to 17, wherein said solidtumour is malignant.
 20. The method or use according to claim 19,wherein said malignant solid tumour is metastatic.
 21. The method or useaccording to any one of claims 18-20, wherein said solid tumour is acentral nervous system tumour, retinoblastoma, neuroblastoma, paediatrictumour, head and neck cancer such as squamous cell cancer, breast andprostate cancer, lung cancer, kidney cancers, such as renal celladenocarcinoma, oesophagogastric cancer, hepatocellular carcinoma,pancreaticobiliary neoplasia, such as adenocarcinomas and islet celltumours, colorectal cancer, cervical cancer, anal cancer, uterine orother reproductive tract cancer, urinary tract cancer, such as of theureter or bladder, germ cell tumour such as a testicular germ celltumour or ovarian germ cell tumour, ovarian cancer, such as an ovarianepithelial cancer, carcinoma of unknown primary, human immunodeficiencyassociated malignancy, such as Kaposi's sarcoma, lymphoma, leukemia,malignant melanoma, sarcoma, endocrine tumour, such as of the thyroidgland, mesothelioma or other pleural or peritoneal tumour,neuroendocrine tumour or carcinoid tumour.
 22. The method or useaccording to any one of claims 1 to 21, wherein said cellular toxin iseither a cytostatic or a cytocidal agent.
 23. The method or useaccording to claim 22, wherein said agent is selected from ActinomycinD, Adriamycin, Arsenic Trioxide, Asparaginase, Bleomycin, Busulfan,Camptosar, Carboplatinum, Carmustine, Chlorambucil, Cisplatin,Corticosteroids, Colicheamicin, Cyclophosphamide, Daunorubicin,Docetaxel, Doxorubicin, Epirubicin, Etoposide, Fludarabine,Fluorouracil, Gemcitabina, Gemcitabine, Gemzar, Hydroxyurea, Idarubicin,Ifosfamide, Irinotecan, Lomustine, Melphalan, Mercaptomurine,Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin, Paclitaxel,Platinol, Platinex, Procarbizine, Raltitrexeel, Rixin, Steroids,Streptozocin, Taxol, Taxotere, Thioguanine, Thiotepa, Tomudex,Topotecan, Treosulfan, Trihydrate, Vinblastine, Vincristine, Vindesine,Vinorelbina, Vinorelbine, duanomycin, dactinomysin, esorubisin,mafosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, MitomycinC, mithramycin, prednisone, hydroxyprogesterone, testosterone,tamoxifen, dacarbazine, hexamethylmelamine, pentamethylmelamine,amsacrine, chlorambudil, methylcyclohexylnitrosurea, nitrogen mustards,Cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, deoxyco-formycin, 4-hydroxyperoxycyclophosphoramide,5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), colchicine,trimetrexate, teni-poside, and diethylstilbestrol.
 24. The method or useaccording to claim 22, wherein said agent is selected from a DNAdamaging agent (eg. nucleophosmin or agents which induce cellular damageas part of a synergistic process with another agent), a catalyticantibody, prodrugs, CHK1/2 inhibitor (e.g. CBP-501 or AZD7762), histonedeacetylase inhibitor (e.g. vorinostat), tumour necrosis factor relatedapoptosis inducing ligand or BH3 mimetic (e.g. ABT737), small moleculeinhibitors such as the tyrosine kinase inhibitors imatinib mesylate,gefitinib, erlotinib, and monoclonal antibodies (e.g. rituximab ortrastuzumab).
 25. The method or use according to claim 22, wherein saidagent is a molecule which functions as an RNA interference mechanism.26. The method or use according to claim 25 wherein said molecule is anRNA oligonucleotide.
 27. The method or use according to claim 26,wherein said RNA oligonucleotide is long double stranded RNA (dsRNA),hairpin double stranded RNA (hairpin dsRNA), short interfering RNA(siRNA), short hairpin RNA (shRNA), micro RNA/small temporal RNA(miRNA/stRNA), miRNAs which mediate spatial development (sdRNAs), thestress response (srRNAs) or cell cycle (ccRNAs), RNA oligonucleotidesdesigned to hybridise and prevent the functioning of endogenouslyexpressed miRNA or stRNA or exogenously introduced siRNA.
 28. The methodor use according to any one of claims 1 to 27, wherein said particle orstabiliser is coupled to a ligand, which ligand is directed to saidtumour.
 29. The method or use according to any one of claims 1 to 28,wherein said particulate material is administered prior to the cellulartoxin.
 30. The method or use according to any one of claims 1 to 28,wherein said particulate materials and said cellular toxin areadministered simultaneously.